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Infection and Immunity, February 2008, p. 717-725, Vol. 76, No. 2
0019-9567/08/$08.00+0     doi:10.1128/IAI.00974-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Global Transcriptional Profile of Mycobacterium tuberculosis during THP-1 Human Macrophage Infection{triangledown} ,{dagger}

Patricia Fontán,1 Virginie Aris,2 Saleena Ghanny,2 Patricia Soteropoulos,2 and Issar Smith1*

TB Center,1 Center for Applied Genomics, The Public Health Research Institute, UMDNJ-New Jersey Medical School, 225 Warren Street, Newark, New Jersey 07103-35352

Received 17 July 2007/ Returned for modification 25 September 2007/ Accepted 27 November 2007


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ABSTRACT
 
During lung infection, Mycobacterium tuberculosis resides in macrophages and subverts the bactericidal mechanisms of these professional phagocytes. Comprehension of this host-pathogen relationship is fundamental for the development of new therapies to cure and prevent tuberculosis. In this work, we analyzed the transcriptional profile of M. tuberculosis infecting human macrophage-like THP-1 cells in order to identify putative bacterial pathogenic factors that can be relevant for the intracellular survival of M. tuberculosis. We compared the gene expression profile of M. tuberculosis H37Rv after 4 h and 24 h of infection of human macrophage-like THP-1 cells with the gene expression profile of the strain growing exponentially in broth cultures. We found 585 genes expressed differentially by intracellular M. tuberculosis. An analysis of the gene expression profile of M. tuberculosis inside THP-1 cells suggests the perturbation of the cell envelope as a major intracellular stress inside THP-1 macrophages.


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INTRODUCTION
 
Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that together with human immunodeficiency virus (HIV) and malaria, is one of the main causes of mortality due to an infectious agent (31). According to the WHO, one-third of the world's population is infected asymptomatically with M. tuberculosis, representing a large reservoir of infection (11). To block further transmission and reactivation in the already-infected population, it is necessary to develop improved intervention strategies that require a better understanding of the host-pathogen interaction.

Infection of a mammalian host by M. tuberculosis usually occurs by the aerosol route, and the lung is typically the principal organ affected. The bacteria initially reside in alveolar macrophages (18), where they are usually able to replicate. In order to identify M. tuberculosis components that may be responsible for successful bacterial intracellular survival, many individual genes whose expression levels are up regulated by the microorganism inside the phagosome have been analyzed (16). DNA microarray technology has made it possible to analyze the M. tuberculosis global transcriptional response to different stimuli. Experiments have been carried out in broth culture, using conditions that may mimic the macrophage environment (i.e., low pH, cell wall stress, starvation, hypoxia, heat shock, etc.) in resting or activated mouse macrophages, and in vivo, using the mouse lung model of infection. The results of these studies have recently been reviewed (2, 29). The complete gene expression profile of M. tuberculosis growing in mouse macrophages was defined by Schnappinger et al. (52) and more recently by Rachman et al. (44). These analyses indicate that inside the mouse macrophage phagosome, M. tuberculosis has to face a DNA- and cell envelope-damaging environment that is rich in fatty acid and deficient in iron. The transcriptional profile of M. tuberculosis infecting human lungs indicates that the bacteria regulate genes involved in the evasion of the immune system (45). A similar analysis of M. tuberculosis in human monocyte-derived macrophages after 7 days of infection suggested the relevance of bacterial genes involved in transcriptional regulation (8). In addition, M. tuberculosis genes that are essential for the survival of bacteria in mouse macrophages and in mouse lungs have been identified by using the transposon site hybridization technique (46) and designer arrays for defined mutant analysis (30).

In this work, we analyzed the gene expression profile of M. tuberculosis strain H37Rv infecting human macrophage-like THP-1 cells. These cells treated with phorbol-myristate acetate (PMA) differentiate into mature macrophages, providing a good model for analyzing the interaction of M. tuberculosis with primary macrophages in terms of receptor expression, bacterial uptake, survival, and replication (59). It has been demonstrated that after infection with M. tuberculosis, THP-1 cells, in a manner similar to that of monocyte-derived macrophages, produce low levels of oxygen radicals and do not produce nitric oxide (55). Moreover, THP-1 cells are a good model to study the effect of M. tuberculosis on apoptosis in human macrophages since they behave in a manner similar to that of primary macrophages in this process (47). Although mouse bone marrow macrophages are widely utilized as a model of infection, M. tuberculosis is a human pathogen and the THP-1 cell model can provide valuable data to better understand the interaction of M. tuberculosis with human macrophages. Furthermore, the use of a cell line may overcome some difficulties that the use of experimentally infected human primary cells presents, i.e., genetic variability and other differences from donor to donor may affect data reproducibility and may consequently require a large number of samples to analyze. In addition, the comparison of M. tuberculosis transcriptome results for THP-1 cells with those obtained for other macrophage models can provide valuable information to improve our knowledge of M. tuberculosis macrophage infection.


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MATERIALS AND METHODS
 
Bacterial cultures. M. tuberculosis strain H37Rv (American Type Culture Collection) was used for all the experiments. Bacterial stocks were maintained at –80°C in 7H9 (Middlebrook 7H9 [Difco]) supplemented with 0.5% bovine serum albumin, fraction V (Boehringer Mannheim), 0.2% glucose, and 0.085% NaCl, 0.2% glycerol, and 0.1% Tween 80. Aliquots of bacteria were thawed and cultured on supplemented 7H10 solid medium. For the preparation of liquid cultures, bacteria growing on 7H10 solid medium were suspended in supplemented 7H9 liquid medium at an optical density (OD) at 540 nm of 0.05, which is equivalent to 5 x 106 CFU per milliliter. Liquid cultures were grown in plastic roller bottles in a roller apparatus up to an OD at 540 nm of 0.2 to use for macrophage infection and for M. tuberculosis RNA extraction.

Macrophage cultures. The THP-1 human monocytic cell line was obtained from the American Type Culture Collection. Cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 0.45% glucose, 0.15% sodium pyruvate, and 4 mM L-glutamine. Cultures were maintained at a density not greater than 106 cells/ml.

Macrophage infections for bacterial RNA preparation. Ten 175-cm2 flasks were treated with 15 ml 0.2% sterile gelatin overnight at 4°C to improve the adherence of THP-1 cells. Prior to use, the gelatin was poured off and the flasks were allowed to dry. Flasks were seeded with 100 ml of 106 cells/ml. THP-1 cells were differentiated with 40 nM PMA overnight at 37°C, 5% CO2, and 95% humidity. M. tuberculosis cultures were grown exponentially in 7H9 liquid medium to an OD of 0.2 (2 x 107 CFU/ml) for the infection. An appropriate volume of this culture was suspended in RPMI with 20% FCS to obtain a bacterial suspension of 2 x 106 CFU/ml. A total of 100 ml of bacterial suspension was added to each flask at a multiplicity of infection of 2, and macrophages and bacteria were incubated for 2 h. After incubation, macrophages were washed twice with 50 ml of warm phosphate-buffered saline (PBS) and 100 ml of RPMI plus 20% FCS was added to each flask. At different time points, cells from five flasks were processed for bacterial RNA extraction as described below. For CFU determinations, a flask from each experiment was sampled for bacteria in the supernatant. Then cells were lysed by treating the monolayer with 10 ml of 0.05% sodium dodecyl sulfate (SDS), and serial dilutions were plated in 7H10 medium.

Bacterial and macrophage RNA extraction and purification. (i) M. tuberculosis RNA from in vitro cultures. A total of 40 ml of exponentially growing bacterial liquid cultures (2 x 107 CFU/ml) were centrifuged, and cell pellets were suspended in 1 ml TRI reagent (Molecular Research Center, Cincinnati, OH) and transferred immediately to a 2-ml screw-cap microcentrifuge tube containing 0.5 ml zirconia beads (0.1-mm diameter, BioSpec Products, Inc., OH). We disrupted the samples by two 1-min pulses in a bead beater, keeping the samples on ice for 2 min between pulses. Liquid was removed from the beads, incubated at room temperature for 10 min, and centrifuged for 10 min at 12,000 x g. The supernate was transferred to a clean tube, 100 µl BCP Reagent (Molecular Research Center) was added to 1 ml samples in TRI, and then the tubes were shaken vigorously for 15 s, incubated for 10 min at room temperature, and then centrifuged for 15 min at 12,000 x g. The supernate was recovered and precipitated with 600 µl isopropanol. After being washed with 75% ethanol, RNA pellets were resuspended in 30 to 100 µl of diethyl pyrocarbonate-treated H2O and then treated with RNase-free DNase (Ambion) for 30 min at 37°C. After DNase treatment, samples were further purified by using RNeasy columns (Qiagen). Purified RNA was kept at –80°C until further use.

(ii) M. tuberculosis RNA from macrophage cultures. Macrophages infected with M. tuberculosis were lysed with 20 ml per 175-cm2 flask of guanidinium thiocyanate-based buffer (25 mM sodium citrate-4 M guanidine thiocyanate-0.5% N-lauryl sarcosine-0.125 M mercaptoethanol-0.5% Tween 80, pH 7.0) (6), and eukaryotic DNA was sheared by homogenization of the samples for 5 min with a VirTis homogenizer. Samples were centrifuged for 30 min at 4,000 x g, and bacterial pellets were washed with 10 ml of guanidinium thiocyanate-based buffer and centrifuged for 15 min at 4,000 x g. Finally, bacterial pellets were suspended in 1 ml of TRI and processed as described above.

DNA micrroarrays. M. tuberculosis DNA microarrays were printed at the Center for Applied Genomics (www.cag.icph.org) at the Public Health Research Institute. The M. tuberculosis microarray consists of 4,295 70-mer oligonucleotides, representing the 3,924 predicted open reading frames of the H37Rv strain (www.sanger.ac.uk), with an additional 371 probes designed to detect sequences present in the CDC1551 strain (www.tigr.org). The arrays were prepared by spotting oligonucleotides (Tuberculosis Genome Set, version 1.0; Operon Biotechnologies) onto poly-L-lysine-coated glass microscope slides by using a GeneMachines OmniGrid 100 arrayer (Genomic Solutions) and SMP3 pins (TeleChem). The complete gene list and array layout can be found at www.cag.icph.org/downloads_page.htm). M. tuberculosis RNA was obtained from H37Rv strain cultured exponentially in 7H9 or recovered at 4 h or 24 h after macrophage infections as described above. A total of 0.5 to 1 µg of total RNA from each sample was used for each microarray. Five (24 h) or six (4 h) biological replicates were analyzed for each time point. Total RNA was reverse transcribed into cDNA by using SuperScript II RT in the presence of cyanine 3 or cyanine 5 dUTP (PerkinElmer) by a modification of the procedure described by Voskuil et al. (63). This was accomplished by using a reaction mixture with a final concentration of 0.17 µg/µl random hexamers (Integrated DNA Technologies); 0.96x first-strand buffer (Invitrogen); 9.6 mM dithiothreitol (Invitrogen); 0.44 mM dATP, dCTP, and dGTP (Invitrogen); 0.02 mM dTTP (Invitrogen); 0.06 mM cyanine 3 or 5 dUTP (PerkinElmer); and 9.4 U SuperScript II (Invitrogen). The combination of the reaction mixture and total RNA was incubated for 10 min at 25°C, followed by 10 min at 42°C. The labeled cDNA probes were then purified and concentrated by using a Microcon YM10 filter (Millipore) and Tris-EDTA buffer, pH 8.0 (Ambion). Slides were blocked prior to hybridization by using bovine serum albumin. The purified cDNA probes were added to the arrays, together with a hybridization solution mix, resulting in a final concentration of 0.5 µg/µl tRNA (Invitrogen), 2.0x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) (Ambion), 0.25% formamide (Sigma), and 0.1% SDS (Ambion). The probes were covered by a flat 22- by 22-mm coverslip (Corning) and hybridized in sealed hybridization chambers (GeneMachines) for 16 h in a 50°C water bath. The detailed labeling and hybridization protocol can be obtained at www.cag.icph.org/downloads_page.htm. The slides were scanned using an Axon 4000B scanner, and the images were processed using GenePix 5.1. Data were filtered by removing all spots that were below the background noise or flagged as "bad." Spots were considered to be below the background noise if the sum of the median intensities of the two channels was less than twice the highest mean background of the chip. Chips were normalized by the print-tip Lowess method (16a). A false discovery rate of less than 2% and a regulation of at least 1.8-fold were used as criteria to consider a gene differentially regulated. False discovery rates were determined by using the Significance Analysis of Microarray program (61). Data were deposited in the Gene Expression Omnibus repository (see below). The hierarchical cluster of the DNA microarray results was performed with the Euclidian distance measure and the average linkage method (17) (see Fig. S1 in the supplemental material).

The determination of significant clusters of regulated genes in the M. tuberculosis chromosome was performed in a manner similar to that in an analysis described previously (52). The number of regulated genes within 10 genes upstream and 10 genes downstream of each evaluated position was determined and divided by 20 (the number of genes in the segment of the chromosome being analyzed). Each result number was divided by the ratio of the number of regulated genes minus one to the number of genes in the chromosome minus one. The resulting quotients are the relative densities and were calculated independently for induced and repressed genes. Values for repressed genes were multiplied by –1. To derive the 5% null hypothesis cutoff value, 200 datasets were generated in which the positions of regulated genes were randomly selected from 3,924 possible positions. The maximum relative density was calculated for each of the 200 datasets, and the 10th largest of these numbers was used as the 1% cutoff value. Relative densities greater than the 1% cutoff value and with more than five differentially regulated genes were defined as "clusters." To identify significant gene groups commonly regulated by stress conditions in vitro as opposed to random coincidences, we used the Expression Analysis Systematic Explorer algorithm (25) from MeV software (50) to calculate the Fisher exact test value for the probability of common occurrences. The gene ontology for M. tuberculosis was downloaded from the TubercuList website (http://genolist.pasteur.fr/TubercuList).

Quantitative RT-PCR and real-time PCR with SYBR green. Reverse transcription (RT) and PCR primers were designed by using the software OLIGO 6.6 (Molecular Biology Insights, Cascade, CO) and purchased from Integrated DNA Technologies (Coralville, IA). For reverse transcription, 50 ng of RNA were used. After denaturation at 94°C for 2 min, annealing between the RNA and the antisense primers was carried out for 3 min at 65°C, followed by 5 min at 57°C. Subsequently, 12 µl of the annealing mixture was added to the RT mix containing Transcriptor RT polymerase (Roche). Samples were incubated for 60 min at 65°C, heated to 95°C for 1 min, and then chilled on ice. Control samples, not treated with Transcriptor RT polymerase, were also prepared. These template samples for PCRs were then diluted with H2O and stored at –20°C. PCR conditions were identical for all reactions. The 25-µl reaction mixtures consisted of 1x DNA polymerase buffer, 2 mM MgCl2, 0.25 mM each deoxynucleoside triphosphate, 1.25 U AmpliTaq Gold polymerase (PerkinElmer), 0.5 µM of each primer, 0.1 µl of SYBR green, and 10 µl of the template. After 10 min at 94°C to activate the DNA polymerase, a set of 35 cycles was run. The denaturation step (20 s) was at 94°C, and the annealing was at 65°C (45 s). The reactions were carried out in sealed tubes in an Mx4000 spectrofluorometric thermal cycler (Stratagene). Fluorescence was measured during the annealing step and plotted automatically for each sample. In order to obtain a standard curve for the RT-PCR, PCR was performed with each primer set by using different amounts of chromosomal DNA and these reactions were performed at the same time as the RT-PCR. These standard curves were used to calculate the amount of cDNA for each gene present in the different samples.

Microarray data accession number. Data were deposited in the Gene Expression Omnibus repository under the accession number GSE6209.


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RESULTS AND DISCUSSION
 
M. tuberculosis infection in THP-1 cells. Using PMA-differentiated THP-1 cells as a model of primary macrophages, we analyzed the global gene expression of M. tuberculosis strain H37Rv after 4 h and 24 h of infection. Cells were infected at a multiplicity of infection of 2. After 2 h in contact with the macrophages, extracellular bacteria were removed by two washes with warm PBS. Approximately 30% of the initial bacterial inoculum (7.25 x 105 ± 3.54 x 104) was associated with the macrophages, and approximately 2% of the initial bacteria inoculum remained in the supernatant (Fig. 1). After 24 h of infection, M. tuberculosis was able to survive and replicate inside THP-1. However, an accurate quantification of bacteria inside macrophages is difficult and we speculated that the dramatic change of 1 log in the number of CFU that we have observed in this work and also previously (15, 33, 66) is mainly due to the disruption of small bacterial clamps. The macrophages were 98% viable during the 24 h of infection, and less than 10% of cells were detached after 24 h (data not shown).


Figure 1
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  		FIG. 1. M. tuberculosis infection of THP-1 macrophages. Macrophages were infected with the H37Rv M. tuberculosis strain. The bacterial inoculum (black bar) was left in contact with the macrophages for 2 h, and the noninternalized bacteria were removed by two washes with warmed PBS. Intracellular bacteria were quantified at the 0-h time point (gray-black bar) and the 24-h time point (gray bar). Bacteria in the supernate were quantified at the 0-h time point (white-gray bar) and the 24-h time point (white bar). The figure shows the means ± standard deviations (error bars) of three independent experiments.

Distribution of M. tuberculosis genes differentially regulated inside THP-1 cells. There were 585 genes of M. tuberculosis strain H37Rv differentially regulated after 4 h and/or 24 h of THP-1 infection. Of these, 379 genes were up regulated (80 at 4 h, 122 at 24 h, and 177 at 4 h and 24 h) and 206 genes were down regulated (63 at 4 h, 62 at 24 h, and 81 at 4 h and 24 h). For a complete list of regulated genes, see Tables S1 and S2 in the supplemental material. An analysis of differential gene expression between 4 h and 24 h shows that 17 genes were significantly up regulated at 4 h but not at 24 h and 7 genes were significantly up regulated at 24 h, but not at 4 h (Table 1). Particularly, several of the genes up regulated at 4 h are in the mymA operon (Rv3083 to Rv3089), whose genes have been observed to be up regulated when M. tuberculosis was exposed to a low pH in vitro (19), and these genes have been associated with the restructuring of the bacteria cell envelope (54). Recently, it has been demonstrated that an M. tuberculosis mutant strain lacking the mymA operon has an altered cell wall structure, is more susceptible to antimycobacterial drugs at a low pH, and is attenuated in macrophages, although the mutant is not defective for growth under low-pH conditions (10, 53).


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  		TABLE 1. M. tuberculosis genes differentially regulated between 4 and 24 h after THP-1 cell infection

The distribution of regulated genes, after the infection of THP-1 cells, into different functional categories indicated that a significant number of genes involved in lipid metabolism were up regulated at 4 h and 24 h, genes in the category of intermediary metabolism were up regulated in a statistically significant number at 4 h, and information pathway genes were significantly up regulated at 24 h. The increase in the number of up-regulated genes involved in lipid metabolism reinforces the general idea that fatty acids are the main intracellular carbon source for M. tuberculosis, and the changes in lipid metabolism are also probably associated with the maintenance of the lipid-rich envelope. Among the down-regulated genes, a group of PE/PPE proteins was significantly repressed at 4 h and 24 h (Fig. 2). The differential regulation of expression for genes encoding PE/PPE family proteins inside macrophages has previously been observed and may be associated with the restructuring of the bacterial cell envelope (12, 14).


Figure 2
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  		FIG. 2. Distribution of M. tuberculosis genes in functional categories. Genes differentially regulated at 4 h and 24 h after infection of macrophages were distributed in functional categories according to TubercuList (http://genolist.pasteur.fr/TubercuList/). Significant differences are indicated by asterisks. *, P < 0.05 (Fisher exact test). Shown are results for genes up regulated at 4 h (black bars), genes down regulated at 4 h (white bars), genes up regulated at 24 h (gray-black bars), and genes down regulated at 24 h (gray bars).

The transcriptional response of M. tuberculosis inside THP-1 macrophages was compared to the global gene expression of M. tuberculosis in vitro under stress conditions like low pH (19), starvation (4), heat shock (57), hypoxia (64), low iron (48), cell wall stress induced by SDS treatment (33), oxidative stress induced by treatment with diamide (32), and the transcriptome of M. tuberculosis inside resting murine bone marrow macrophages (BMM) (52). Figure 3 shows the statistically significant number of common genes differentially regulated in a given stress condition and in THP-1 in relationship to the total number of genes regulated in both conditions. For the identity of these genes, see Fig. S2 in the supplemental material. The statistically significant number of genes differentially regulated in THP-1 and under the different stresses indicated that these in vitro conditions can mimic some of the expected properties of the intraphagosome environment. It is worth noting that genes up regulated under iron limitation are down regulated inside macrophages at 4 h and 24 h after infection. We observed a significant number of M. tuberculosis genes regulated in both resting murine BMM and PMA-differentiated THP-1 cells. From the list of 68 M. tuberculosis genes that were found to be regulated, particularly after the infection of gamma interferon-treated BMM (52), we identified 11 genes that were also up regulated after infection of THP-1: Rv0080, Rv0081, Rv2028c, pfkB, Rv2030c, acr, acg, Rv2626c, Rv3133c, fadE24, and fadE23. Differences in the correlation between both gene expression analyses could be attributed to the macrophages used.


Figure 3
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  		FIG. 3. Comparison of M. tuberculosis transcriptome in THP-1 cells with the M. tuberculosis transcriptome in different stress conditions in vitro and in BMM. Genes differentially regulated in THP-1 were compared with genes differentially regulated after different treatment in vitro and after M. tuberculosis infection of BMM. The figure shows the number of common differentially regulated M. tuberculosis genes in different stress conditions, BMM, and THP-1 cells. Shown are results for genes down regulated at 4 h in THP-1 (white bars), genes up regulated at 4 h in THP-1 cells (gray bars), genes down regulated at 24 h in THP-1 cells (gray-black bars), and genes up regulated at 24 h in THP-1 cells (black bars). A P value of <0.02 was significantly different (Fisher exact test). For the identities of the genes, see Fig. S2 in the supplemental material.

To determine the existence of groups of genes from regions in the M. tuberculosis chromosome that are regulated simultaneously, we performed a determination of significant clusters of regulated genes. This statistical analysis indicated that 158 differentially regulated genes at 4 h and 24 h are grouped in nine clusters along the chromosome. Table 2 shows the summary of the cluster analysis, and for the complete list of genes in each cluster, see Fig. S3 in the supplemental material. There was only one cluster of genes down regulated at 4 h of infection, and two clusters were down regulated at 24 h of infection. The cluster of down-regulated genes at 4 h included many unknown proteins and several members of the PE/PPE family of M. tuberculosis proteins. This cluster overlaps with one of the clusters of down-regulated genes at 24 h of infection and includes a putative operon that contains a secretion system of the ESAT-6-like family. The second cluster of genes down regulated at 24 h included genes probably involved in intermediary metabolism and cell wall processing. We also identified four clusters of up-regulated genes after 4 h of infection. One of these clusters included genes related to intermediary metabolism and conserved hypothetical proteins. Many of these genes are annotated as coding for hydrogenases, and they are organized in a putative transcriptional unit. The up regulation of this cluster was also observed at 24 h after infection. Since it has been postulated that hydrogenases play a role in membrane-linked energy conservation through the generation of a proton motive force (62), the use of hydrogenases may be a mechanism for M. tuberculosis to conserve energy under stress. Moreover, the up-regulated expression of hydrogenases induced by low pH and anaerobiosis has previously been described for Escherichia coli by Hayes et al. (21). These authors suggested that besides being a mechanism to gain energy, hydrogenases might consume protons to reverse acidification.


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  		TABLE 2. Clusters of regulated genes in the chromosome of M. tuberculosis

The second cluster of up-regulated genes at 4 h of infection includes genes related to lipid and intermediary metabolism. This cluster includes the mymA operon described above. The other two clusters of genes up regulated at 4 h are composed mainly of genes annotated as encoding conserved hypothetical proteins and intermediary metabolism components. The second cluster of genes up regulated at 24 h corresponded to the phthiocerol dimycocerosate locus, whose products are structural components of the M. tuberculosis cell envelope involved in the virulence of this microorganism (7). This analysis indicated that the relevant changes in the expression levels of clusters of genes related to energy and cell envelope remodeling occurs after M. tuberculosis infection of THP-1 cells. Of particular interest is the lag in expression of the virulence-related phthiocerol dimycocerosate locus.

Functional classification of M. tuberculosis genes differentially regulated inside THP-1: metabolism, respiration, and energy. The genes coding for enzymes of the tricarboxylic acid gltA1, gltA2, can, and sucA were up regulated by M. tuberculosis during THP-1 infection. It has recently been determined that sucA encodes an alpha-ketoglutarate decarboxylase that allows bacteria to perform a bypass from alpha-ketoglutarate to succinate (60). Both gluconeogenesis and glycolysis seem to be functional during infection, since the genes encoding phosphoenolpyruvate, carboxykinase (pckA), and phosphofructokinase II (pfkB) are up regulated, as has previously been observed (15, 52).

Sulfate is an important cell component that is taken up by a sulfate permease. The sulfate can be activated to adenosine 5'-phosphosulfate and further phosphorylated to 3'-phosphoadenosine 5'-phosphosulfate by two ATP sulfurylases, CysD and CysN. The genes that encode these enzymes were up regulated in macrophages at 4 h after infection (this work) and under SDS and oxidative stress (33). 3'-Phosphoadenosine 5'-phosphosulfate can be incorporated into the bacterial sulfolipids that are components of the cell envelope, and it has been postulated that these molecules may contribute to bacterial virulence by interfering with phagolysosome fusion (43).

We observed that the expression of the gene ppa that encodes a pyrophosphatase was up regulated by M. tuberculosis in human macrophages. In growing bacterial cells, the synthesis of macromolecules like DNA, RNA, and proteins generates PPi that is further catabolized by an inorganic pyrophosphatase (Ppa). In E. coli, this enzyme is produced constitutively and is essential for growth (9). Up regulation of the inorganic pyrophosphatase (ppa) gene has been observed before in intracellular-growing Legionella pneumophila. In this microorganism, the up-regulated expression of ppa has been correlated with the higher growth rate of these bacteria in vivo compared with the growth in vitro and the concomitant need of macromolecule synthesis (1). In M. tuberculosis, up regulation of the gene ppa may be related to an intracellular condition of phosphate starvation, as postulated recently by Rachman et al., who also observed this gene up regulated by M. tuberculosis infecting mouse macrophages (44).

Finally, many genes required for the synthesis of amino acids, purines, and pyrimidines were up regulated, indicating that bacteria are not able to obtain these components from the phagosome.

Iron metabolism. Although it has been postulated that macrophages are low-iron environments for M. tuberculosis (20), we did not observe a significant up regulation inside the macrophages of the mbt genes encoding the M. tuberculosis siderophores responsible for iron uptake. Indeed bfrA, a gene that encodes a putative iron storage protein and requires high levels of iron to be expressed (48), was up regulated during M. tuberculosis infection in THP-1 cells. Moreover, many of the genes included in the cluster of genes down regulated at 4 h in THP-1 (Rv0283 to Rv0292) are up regulated in low-iron conditions (48). These genes likely would be up regulated in THP-1 macrophages if the intracellular environment was iron deprived. It has also recently been demonstrated that the iron levels are high in phagosomes of inactivated macrophages infected with M. tuberculosis (65). In gamma, interferon-activated mouse macrophages, the genes involved in iron uptake that are under IdeR repression are up regulated, suggesting a low availability of iron in this condition. Nevertheless, it has also been postulated that in activated macrophages, the up regulation of these genes is due to the inactivation of IdeR by nitric oxide, not by a low-iron concentration (52). In a recent work by Rachman et al., it has also been observed that the transcriptional response of M. tuberculosis to a low-iron environment correlates particularly with the bacteria, transcriptional profile inside activated macrophages (44).

Lipid metabolism. A significant number of M. tuberculosis genes differentially regulated in macrophages in our array were associated with lipid metabolism (Fig. 2). The isocitrate lyase (icl) of M. tuberculosis is up regulated in bacteria infecting THP-1 macrophages. This enzyme is a key component of the glyoxylate cycle that allows M. tuberculosis to obtain a source of energy from fatty acids broken down to acetyl coenzyme A. Isocitrate lyases have been shown to be essential for the pathogenesis of this microorganism and its intracellular survival in mice (37). M. tuberculosis has 20 genes coding for cytochrome P450s, four of which were up regulated in bacteria infecting THP-1 cells. The function of these cytochromes has been related to drug activation, and it has been suggested that in M. tuberculosis, the function can be related to lipid metabolism (36).

The expression of genes from the mymA operon was up regulated by exposure of M. tuberculosis to low pH (19) in vitro and also in THP-1 cells after 4 h of infection. Most of the genes in this regulon have been related to fatty acid metabolism. These observations may indicate that the switch of M. tuberculosis to fatty metabolism can be triggered by changes in the intracellular pH of macrophages.

Cell envelope and secretion. M. tuberculosis genes involved in phthiocerol dimycocerosate synthesis and transport to the cell surface, such as drrB, ppsA, ppsB, and pks11, were up regulated by the bacteria inside THP-1 cells. The genes coding for the polyketide synthases, pks10, pks9, and other pks-associated proteins of unknown functions as well as the mycolic acid methyltransferase umaA1, were differentially expressed. Moreover, cpsY and rmlB2 genes were up regulated in macrophages. These genes encode UDP-glucose-4-epimerases that are related to cell wall restructuring, since they are involved in the synthesis of galactofuran, which is essential for the linking of peptidoglycan and mycolic acid (67). We also found many M. tuberculosis genes differentially regulated during infection of THP-1 cells, such as ftsW, ftsE, ftsX, ftsH, murC, and murG, that in E. coli are annotated to be associated with cell division and peptidoglycan assembly (41, 51).

Rv0986, which codes for a component of an ABC transporter, was one of the most up-regulated genes in our experiments. The screening of a mutant library to look for trafficking-deficient Mycobacterium bovis BCG indicated that bacteria with mutations in Rv0986 are inhibited in the ability to avoid trafficking to the phagolysosome (42). More recently, it has been demonstrated that Rv0986 and Rv0987 are involved in bacterial adherence to macrophages (49).

The Sec-independent protein translocase (tatA) is encoded by the gene Rv2094c, which was up regulated in M. tuberculosis inside THP-1 cells. It has previously been demonstrated that E. coli strains with mutations in components of the Tat pathway are defective in the integrity of the cell envelope (26). The relevance of the twin arginine translocase in bacterial pathogenesis has been well characterized for Pseudomonas aeruginosa (40). Recently, it has been demonstrated that the Tat translocation pathway is required in Mycobacterium smegmatis and M. tuberculosis for the export of β-lactamases (34). M. smegmatis strains with mutations in the tatA or tatC genes grew poorly in vitro and exhibited high sensitivity to SDS. In our array data, we observed the modulation of ESAT-6-like proteins, such as esxM, esxG, and esxS. Interestingly, esxG (Rv0287) and cfp-7 (Rv0288) are down regulated, together with a cluster of another five genes in the region of Rv0280 to Rv0286 that are annotated as PE, PPE, and membrane proteins. Possibly, these genes code for a secretion system similar to that of ESAT-6, but the meaning of the down regulation of its expression in macrophages and its role in the pathogenicity of M. tuberculosis remains to be studied. The expression of the gene coding for the secreted protein PirG was differentially regulated in our array data. PirG (Erp) is part of a family of exported proteins that have a common repetitive motif, and it has previously been described as a virulence factor. The erp knockout mutant of M. tuberculosis displays very low levels of multiplication both in macrophage cell lines and in an in vivo mouse model of infection; however, its function is not known (13).

Besides the genes that we have stressed above, we also found differentially regulated genes that encode 10 lipoproteins, many transporters, and several membrane proteins of unknown functions. All these observations indicate that the remodeling of the cell wall and the secretion of proteins are major mechanisms for M. tuberculosis to adapt to intracellular life.

Transcriptional regulators. In our DNA microarray analysis, the expression of the gene coding for IdeR was up regulated during the infection of THP-1 cells as observed previously (24). IdeR is a regulator that represses the expression of genes related to iron uptake and positively regulates genes that code for iron storage proteins (20). WhiB is a transcriptional factor essential for sporulation in Streptomyces coelicolor. In its genome, M. tuberculosis has several orthologs of the whiB gene (56). In our study, whiB3 was up regulated and whiB2 was down regulated in the intracellular bacteria. WhiB3 binds to RpoV and a mutant of M. tuberculosis strain H37Rv. A disruption of the whiB3 gene shows an attenuated phenotype in mice (58). WhiB3 was also up regulated in mouse macrophages after 6 h of infection (3); this observation, together with the up regulation in THP-1, may indicate a differential regulation of this transcription factor in inactivated macrophages.

The transcriptional regulator Rv3133c (dosR), which has been associated with hypoxia, nitric oxide, and dormancy (64) and more recently with other conditions like oxidative stress (28), was up regulated in THP-1 cells. We also found that intracellular M. tuberculosis up regulated Rv0981 (mprA). This transcriptional regulator has been associated with persistence (68), and it has been demonstrated that it controls the expression of genes that respond to SDS-stress-like sigma factor E and sigma factor B (22). Interestingly, none of the 13 sigma factors of M. tuberculosis are differentially regulated in bacteria growing intracellularly.

Oxidative stress and heat shock. Several antioxidant mechanisms are up regulated when M. tuberculosis enters macrophages. Two highly up-regulated genes in our experiments were AhpC and AhpD. AhpC is the most important component of an NADH-dependent peroxidase and peroxynitrite reductase system, where it directly reduces peroxides and peroxynitrite and is in turn reduced by AhpD and other proteins (23). The up regulation of sodA, which encodes a superoxide dismutase, also indicates that the intracellular bacteria are exposed to oxidative stress. But since katG, which codes for the M. tuberculosis catalase, was not up regulated, probably the detoxification of hydroxyperoxide generated by the superoxide dismutase as well as the one originated by lipid metabolism occurs, as has previously been suggested (27), through the thioredoxin, glutaredoxin, or ahpC system. Besides these pathways, M. tuberculosis has a mycothiol system that replaces the glutathione system present in other bacteria (39). In our study, the expression of the mca gene, which encodes a mycothiol conjugate amidase, was up regulated. It has been shown that Mca is involved in detoxification, and its expression has previously been observed to be up regulated in macrophages (38). On the other hand, the gene coding for mycothiol synthetase and other enzymes related to these mechanisms of detoxification, such as cysteine synthase and myo-inositol-1-phosphate synthase, was repressed, indicating that M. tuberculosis may preferentially utilize the thioredoxin redox system for the detoxification of oxygen metabolites in THP-1 cells. We also observed the up regulation in THP-1 cells of htpx, hspX, and htpG, which encode heat shock proteins. cspA and cspB are genes annotated as coding for cold shock proteins and were differentially expressed in our experiments.

We have validated, by quantitative RT-PCR, the DNA array data for selected M. tuberculosis genes that we found differentially expressed in macrophages and whose role in the intracellular survival of the bacteria is discussed above (Fig. 4).


Figure 4
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  		FIG. 4. Validation of M. tuberculosis gene expression inside THP-1 cells by quantitative RT-PCR. The expression of the indicated M. tuberculosis genes in the intracellular bacteria at 4 h (black bars) and 24 h (gray bars) was compared with that of bacteria growing exponentially in 7H9 broth (A). The expression of each gene in every condition has been normalized to the expression of sigA. Fold induction is expressed as means ± standard deviations (error bars) of ratios from three biological replicates. Data was obtained for these genes by DNA arrays (B).

Concluding remarks. Macrophages are among the most important players in the innate immune defenses that control different infectious processes. However, M. tuberculosis has evolved to subvert the killing mechanisms of these cells and to survive inside the phagosome.

The analysis of the M. tuberculosis transcriptome inside THP-1 cells as a model of primary macrophages indicates that the main transcriptional changes are related to the metabolism of lipids and maintenance and/or remodeling of the cell envelope. Moreover, the transcriptional profile of M. tuberculosis inside primary macrophages does not correspond with a response to a low-iron environment as was previously observed for mouse macrophages (52). The high up regulation of AhpC, the most important component of an NADH-dependent peroxidase, suggests the ability of M. tuberculosis to resist the primary macrophage oxidative burst.

The global transcriptional profile of M. tuberculosis inside THP-1 cells supports previous reports suggesting that fatty acids are the main carbon source for M. tuberculosis inside the macrophage (35) and that M. tuberculosis has the ability to utilize lipids as sources of nutrients and to maintain the lipid-rich envelope (5). Moreover, differential intracellular expression of other molecules like the PE/PPE and the ESAT-6-like families of proteins as well as the TAT system of secretion suggests that after infection of primary macrophages, M. tuberculosis undergoes several changes in secretion and expression of surface components. The ability of M. tuberculosis to survive inside primary macrophages may depend on the differential expression of these bacterial components, and the remodeling of the cell envelope after the infection of a primary macrophage may be an active mechanism of M. tuberculosis to repress the innate immune system.


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ACKNOWLEDGMENTS
 
We acknowledge German Rehren for assistance with the THP-1 cell cultures; Michael Cody and Anthony Galante from the PHRI Center for Applied Genomics for data analysis of DNA microarrays; and Eugene Dubnau, Richard Pine, and Marcelo Nociari for critical reading of the manuscript.

This work was supported by NIH grants (AI-44856 and HL-68513) awarded to I.S.


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FOOTNOTES
 
* Corresponding author. Mailing address: Room W210W, 225 Warren Street, Newark, NJ 07103-3535. Phone: (973) 854-3260. Fax: (973) 854-3101. E-mail: smithis{at}umdnj.edu Back

{triangledown} Published ahead of print on 10 December 2007. Back

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

Editor: F. C. Fang


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Infection and Immunity, February 2008, p. 717-725, Vol. 76, No. 2
0019-9567/08/$08.00+0     doi:10.1128/IAI.00974-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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