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1 Microbiology Division, Central Drug Research Institute, Lucknow, India
2 INSERM U629, Institut Pasteur de Lille, 1 rue du Professor Calmette, F-59019 Lille Cedex, France
Correspondence
B. S. Srivastava
drbrahm{at}gmail.com
C. Locht
camille.locht{at}pasteur-lille.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Tuberculosis is treated by a combination of four drugs which act on different targets. In recent years, Mycobacterium tuberculosis isolates resistant to one or more of these drugs have been spreading, which seriously hampers successful control measures (WHO, 1997
). Novel drugs acting on new targets are therefore urgently needed.
There are several important macromolecules in the mycobacterial cell which are vital for survival of the organism in host cells and progression of the disease (Smith, 2003). These macromolecules may either be constitutively expressed or their expression may be up-regulated within the host. Finding such targets is the key to developing new drugs.
M. tuberculosis infects by aerosol and is ingested by alveolar macrophages. Contrary to most other invading micro-organisms, which are killed within the macrophages, the tubercle bacillus survives and grows in the unfavourable conditions inside the macrophages. It has been reported that alterations in gene expression in the mycobacteria and in the macrophages take place, which allow the mycobacteria to survive (Barker et al., 1998; Dubnau et al., 2002; Hobson et al., 2002). Studies with other pathogens have shown that genes which are up-regulated during infection are often important for pathogenicity (Chiang et al., 1999). Therefore, the identification of mycobacterial genes that are up-regulated in infected macrophages may provide targets for drug design or vaccine development.
Different approaches have been used to identify microbial genes that play a role in the interaction with host cells and thus in virulence (Jacobs et al., 1991). These include signature-tagged transposon mutagenesis (Camacho et al., 1999), in vivo-induced antigen technology (Deb et al., 2002), substractive hybridization (Plum & Clark-Curtiss, 1994), selective capture of transcribed sequences (Graham & Clark-Curtiss, 1999) and in vivo expression technology (IVET; Mahan et al., 1993). Several important genes have been identified in mycobacteria, including hspX (Yuan et al., 1998), and genes encoding isocitrate lyase (McKinney et al., 2000), mycobacterial cell entry protein (Arruda et al., 1993) and glutamine synthetase (Harth & Horwitz, 1999).
Compared to other techniques, IVET has the advantage of identifying bacterial genes in the host environment. It was originally developed to identify virulence genes of Salmonella typhimurium (Mahan et al., 1993) and this elegant approach is being applied now to other pathogenic bacteria. In the present investigation, we have used a modified IVET strategy based on a promoter-trap plasmid vector. The plasmid contains two reporter genes, gfp, encoding green fluorescent protein, and kan, encoding kanamycin resistance, in a transcriptional fusion downstream of a promoter cloning site. An M. tuberculosis promoter library was screened in macrophages on the basis of gfp expression and kanamycin resistance. Expression levels of genes belonging to the identified promoters were then individually confirmed by real-time PCR analysis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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[F– (
80d
lacZ M15) recA1 endAl gyrA96 thi-1 hsdR17 (
) supE44 relA1 deoR (lacIZYA-argF)U169; Invitrogen] and NM522 [F' proAB lacIq lacZ M15/supE thi (lac-proAB) (hsdMS-mcrB)5 (
) McrB–; MBI Fermentas] were used for cloning of the different constructs and the library. Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155, Mycobacterium bovis BCG Pasteur strain (isolate 1173P2; WHO) were employed in this study. E. coli was grown in Luria–Bertani (LB) broth or plated on LB solidified with 1.5 % (w/v) agar. Mycobacteria were grown in liquid Sauton medium (Sauton, 1912), Middlebrook 7H9 medium (Difco) supplemented with ADC (0.2 % glucose, 0.5 % bovine serum albumin fraction V, 0.0003 % beef catalase) or solid Middlebrook 7H10/7H11 agar (Difco)+OADC enrichment (Becton Dickinson). All cultures were grown at 37 °C with or without shaking. Antibiotics were added into the media when required at the following concentrations: streptomycin, 10–25 µg ml–1; kanamycin, 5–25 µg ml–1; cycloheximide, 50 µg ml–1; gentamicin, 50 µg ml–1.
Fluorescence measurement.
Fluorescence was measured by using a FACS Calibur (Becton Dickinson) and the Cell quest software provided by the supplier. The bacterial population was gated by using side and forward scatter parameters and then analysed for fluorescence. The results are expressed as the percentage population fluorescing and the mean fluorescence intensity.
Construction of promoter-trap vector pLL192.
The E. coli–Mycobacterium shuttle vector pMV261 (Stover et al., 1991), containing an E. coli (colE) and a mycobacterial (oriM) origin of replication, as well as a kanamycin resistance gene and the mycobacterial hsp60 promoter, was chosen to construct pLL192, as shown in Fig. 1. The gene encoding streptomycin resistance (Smr) was amplified by PCR from pHP45
(Prentki & Krisch, 1984), by using primers Strep-F1 (5'-AATGGGGTACCGGTGATTGATTGAGC-3') and Strep-R1 (5'-CCGAAACGCGCGAGGCAAGCTTA-3'). The amplified fragment was directly inserted into the PCRII-TOPO vector (TOPO TA cloning; Invitrogen). The Smr gene to be used as a selection marker was then recovered by a BamHI/Asp718 restriction and inserted into pMV261 previously digested with the same enzymes. After transformation of E. coli NM522, Smr clones were isolated. The promoterless gfp gene was amplified by PCR from pJFX2 (Triccas et al., 1999) by using primers GFP Forw2 (5'-GCGGATCCTCTAGATTTAAGAAG-3') and GFPBlunt (5'-TAAGATATCATTTGTATAGTTCATCCATGCC-3') containing a BamHI and an EcoRV site (underlined), respectively. The promoterless kan gene was amplified by PCR from pMV261 by using the primers KanR1 (5'-GGGTCTGACGCTCAGTGGAA-3') and KanBlunt (5'-GGTGTGTTGACCCATATTCAACGGGAA-3'), the latter containing a HincII site (underlined). The two amplified fragments were digested respectively with BamHI/EcoRV and HincII/SpeI and inserted in a single step into the BamHI/SpeI-digested Smr pMV261 derivative. The GFPBlunt and KanBlunt primers were defined such that, after cloning, the start codon of the kan gene overlaps the stop codon of the gfp gene. The final construct, named pLL192, contains an artificial bicistronic operon composed of the promoterless gfp gene, followed by the kan gene. The presence of a unique BamHI site immediately upstream of this transcriptional fusion allows for the insertion of promoter libraries.
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and Baulard et al. (1996), and partially digested with Sau3AI. After agarose gel electrophoresis, fragments of 100–800 bp were purified (QIAquick Gel Extraction Kit; Qiagen) and inserted into dephosphorylated BamHI-digested pLL192. After transformation of E. coli DH5, DNA was extracted from approximately 100 000 recombinant colonies and electroporated into M. bovis BCG (Gene Pulser II; Bio-Rad; 2.5 kV, 25 µF, 800 ). The mycobacteria were then plated onto Middlebrook 7H10 agar containing streptomycin and cycloheximide. More than 10 000 Smr clones with inserts were obtained. Realizing fully that this number may not represent the whole genome, these clones were harvested and pooled in Middlebrook 7H9 medium (Difco) supplemented with 10 % (v/v) ADC. Glycerol (15 %, v/v) was added and the mycobacteria were stored at –80 °C. Before use, the recombinant mycobacterial pool was grown in Sauton medium to mid-exponential phase.
Macrophage infection.
The J774A.1 mouse macrophage cell line was grown in RPMI (Gibco-BRL) or Dulbecco's modified Eagle medium (DMEM; Gibco-BRL) supplemented with 10 % heat-inactivated fetal calf serum (FCS; Gibco-BRL), at 37 °C in a 5 % CO2 humidified atmosphere. The cells were seeded in 24-well cell culture clusters (Corning) (5x105 cells per well in a 1 ml volume) and allowed to adhere overnight. The macrophages were then infected with a mycobacterial suspension at an m.o.i. of 1 : 5. After 4 h of infection, macrophage monolayers were washed several times to remove the remaining extracellular bacteria and the cells were incubated for an additional 24–48 h in fresh medium containing gentamicin (50 µg ml–1) to kill extracellular bacteria. The macrophages were then lysed with PBS/0.05 % SDS. The fluorescence of intracellular bacteria was analysed by flow cytometry and compared to that observed for bacteria incubated under the same conditions but in the absence of macrophages. In the case of the M. tuberculosis promoter library, macrophages were infected with recombinant BCG for 4 h as described above, several washes were performed, and then they were incubated in fresh culture medium with gentamicin and kanamycin (5 µg ml–1) for an additional 24–48 h to select specifically recombinant BCG containing a promoter activated ex vivo, therefore conferring resistance against this antibiotic. A kanamycin concentration of 5 µg ml–1 was selected because more than 80 % killing of BCG harbouring empty pLL192 was caused by 1 µg kanamycin ml–1 in the medium. Macrophages were then lysed with PBS plus 0.05 % SDS. The recovered bacteria were plated onto Middlebrook 7H10 agar plates supplemented with streptomycin and cycloheximide. The growing colonies were then checked individually for their kanamycin resistance in vitro.
RNA isolation and real-time PCR.
For RNA isolation, J774A.1 macrophages were infected with M. tuberculosis or M. bovis BCG at an m.o.i. of 1 : 5. After 4 h of infection, the macrophages were washed twice with DMEM medium without FCS and then incubated in DMEM medium containing FCS and gentamicin (50 µg ml–1). At different time points (24, 36 and 48 h), the cells were harvested, washed with DMEM medium without FCS and then lysed with Tween normal saline containing 0.1 % Tween (w/v) and 0.1 % (w/v) SDS. The debris of lysed macrophages was removed by centrifugation at 200 g. The supernatant containing bacteria and lysate of macrophages was mixed with two volumes of RNAprotect provided in the Qiagen RNAeasy kit. Bacteria were then pelleted by centrifugation at 10 000 r.p.m. (9000 g) for 7 min and the supernatant was discarded. The bacterial pellet was resuspended in TE+lysozyme (10 mg ml–1) and briefly sonicated after adding RLT buffer from the RNAeasy kit. The RNA was then isolated by using the RNAeasy kit and quantified spectrophotometrically. About 5–10 µg RNA was obtained from each macrophage experiment. Using the same protocol, RNA was isolated from in vitro-grown cultures of M. tuberculosis and M. bovis BCG by resuspending the bacteria in TE+lysozyme, sonication and extraction by using the RNAeasy kit. RNA was also extracted from bacteria incubated in DMEM medium containing FCS, to eliminate the effect of serum components and DMEM, and no difference was observed (data not shown). For real-time PCR analysis, each RNA sample was treated with Promega DNase and heat-inactivated according to the manufacturer's instructions. About 200–400 ng RNA was used per reaction. In each set of reactions, 16S rRNA was used for normalization. Each reaction was repeated three times with three independent RNA samples in a Cepheid SmartCycler II. Primers used in real-time PCR are listed in Table 1. A control without reverse transcriptase was included in each set of reactions, and a DNA melting point curve analysis was also done for each sample. Relative expression levels were determined using the 
method of Livak & Schmittgen (2001), assuming that the PCR efficiencies were the same as seen from the slope of the reaction curves. The results were expressed as -fold induction of expression compared to the in vitro control.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This E. coli–Mycobacterium shuttle vector contains two promoterless reporter genes, one encoding a mutated form of the green fluorescent protein (gfp) and the second corresponding to the kanamycin resistance gene (kan). These two genes are organized into a single transcription unit located immediately downstream from a unique BamHI site, allowing for the cloning of promoter libraries. To validate pLL192 as a promoter-trap vector, the gfp : : kan fusion was placed under the control of three different mycobacterial promoters, namely the constitutive hsp60 promoter (Stover et al., 1991), the icl (isocitrate lyase) promoter (McKinney et al., 2000), known to be induced under specific physiological conditions, and the acetamidase gene promoter (Payton et al., 1999), whose induction is acetamide-dependent. The resulting plasmids were electroporated into M. smegmatis mc2155 and M. bovis BCG and analysed for fluorescence and kanamycin sensitivity. Empty pLL192 was used as a negative control. Fluorescence was observed with bacteria harbouring the hsp60 promoter in pLL192 (Fig. 2a) and these bacteria were resistant to up to 25 µg kanamycin ml–1, compared to the negative control for which more than 80 % killing was observed at 1 µg kanamycin ml–1. When the strain containing the pLL192 derivative with the icl promoter was grown in Middlebrook 7H9 medium containing 10 mM acetate or glucose, fluorescence intensity was found to depend on the presence or absence of acetate or glucose (Fig. 2b). Finally, the construct containing the acetamidase gene promoter showed fluorescence intensities proportional to the concentrations of acetamide used in the media (0.2–2.0 mg ml–1; Fig. 2c).
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Kanamycin (5 µg ml–1) was found to kill effectively the intracellular BCG control containing pLL192 without insert. This correlated well with the lack of fluorescence (data not shown). Macrophages infected with BCG control containing pLL192 without insert were grown in the presence or absence of kanamycin. After incubation, the macrophages were lysed and the bacteria were plated onto Middlebrook 7H10 agar with or without kanamycin. Approximately 1.5x104 colonies grew on the plates when the bacteria came from macrophages grown in the absence of kanamycin and plated onto Middlebrook 7H10 agar without kanamycin. However, no colonies were observed for the control BCG after growth in macrophages cultured in the presence of kanamycin.
When BCG cells containing the promoter library were used to infect macrophages, grown in the presence of kanamycin and subsequently plated onto Middlebrook 7H10 agar without kanamycin, 14 colonies were detected. Eight of these failed to grow on Middlebrook 7H10 agar in the presence of kanamycin. These eight clones were then used again to infect macrophages grown with or without kanamycin. No difference in the survival of the BCG clones was observed between these two conditions, confirming that these clones could survive in macrophages in the presence of kanamycin. However, they remained sensitive to kanamycin when grown in vitro on Middlebrook 7H10 agar, suggesting up-regulation of the corresponding promoters in macrophages ex vivo. When analysed for fluorescence, all eight clones were found to fluoresce strongly ex vivo, but showed no or weak fluorescence in vitro, as shown for one example in Fig. 3.
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) to identify the corresponding genes, and they are listed in Table 2. Real-time PCR analysis using RNA isolated at various time points from M. tuberculosis and M. bovis BCG grown in vitro and within macrophages was then carried out to confirm the up-regulation of these genes. The results are shown in Fig. 4(a, b). After normalization to the 16S rRNA, 2- to 40-fold induction of the specific genes was observed both in M. tuberculosis and M. bovis BCG in infected macrophages, compared to in vitro growth. However, some difference in gene expression was observed between M. tuberculosis and M. bovis BCG.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Maurin & Raoult, 2001). The vector was validated by using different known constitutive and inducible promoters. The combination of GFP and selection by kanamycin allowed us to select M. tuberculosis promoters which were activated and up-regulated within macrophages compared to mycobacteria grown in vitro. Vectors combining GFP with the counterselectable SacB protein (Triccas et al., 1999), or kanamycin pre-selection with SacB post-selection (Saviola et al., 2003), were employed to select M. tuberculosis genes expressed within the macrophages. The simultaneous expression of fluorescence and kanamycin resistance phenotypes conferred by pLL192 in the presence of an activated promoter makes the selection process easy in a single step and has the advantage of usability in vivo. In the first round, eight clones with the desired phenotype were identified. The activities of the reporter genes under the control of these promoters were minimal in vitro as judged by weak or absent fluorescence and kanamycin sensitivity on 7H10 agar, but were high within macrophages. This was confirmed and quantified by RT-PCR. In all cases, expression was up-regulated several-fold within macrophages. Expression of PE_PGRS63 was higher in macrophages than during in vitro culture. Within the macrophages, its expression was maximal after 24 h (the first time point) and then decreased. The expression of mce1A was observed within 24 h of infection and was similar in both M. tuberculosis and M. bovis BCG. The presence of mce1A in many species of mycobacteria, including mycobacteria other than tuberculosis-causing species, suggests that it is an important gene for entry and survival (Arruda et al., 1993).
Rv1026 is a conserved hypothetical protein whose exact function is not yet known, but it may be involved in an adaptive response to amino acid starvation and has been classified as an essential gene by Himar1-based transposon mutagenesis of M. tuberculosis (Sassetti et al., 2003). Hence, up-regulation of Rv1026 might reflect the adaptive response to stress within the macrophage.
Acquisition of iron is an essential feature of many bacterial pathogens as iron is required for growth and survival. Mycobacteria have evolved various mechanisms to acquire iron (De Voss et al., 1999), but the role of the potential mycobactin utilization protein ViuB encoded by Rv2895c has not been studied. However, it shows homology to the vibriobactin utilization protein (ViuB) of Vibrio cholerae (Butterton & Calderwood, 1994), and may thus be involved in intracellular removal of iron from iron–mycobactin complexes.
Rv2232 is a putative phosphatase whose function remains to be defined. The expression of Rv2232 has been shown to be up-regulated significantly in SCID mice, but not in BALB/C mice (Talaat et al., 2004), suggesting that this gene has a role in immunocompromised hosts. Rv2231c (CobC) is a putative transferase involved in cobalamin biosynthesis. Using Himar1-based transposon mutagenesis in M. tuberculosis H37Rv, it was demonstrated that Rv2231c corresponds to an essential gene in H37Rv (Sassetti et al., 2003). The role of Rv1635c and Rv0997 is not known.
The deduced functions of selected genes are consistent with conditions encountered within the macrophages. The induction of their expression observed ex vivo may therefore represent a strategy employed by M. tuberculosis to promote the infection and to enhance intracellular survival. This investigation has led to the selection of promoters that are activated over 48 h after the beginning of the infection. Thus it might have facilitated identification of promoters which are induced early and then down-regulated, which is supported by the RT-PCR data. However, the survival of the bacilli and their multiplication inside macrophages might also involve other genes displaying an up-regulated expression during extended periods of time. Therefore, it might be fruitful to subject the promoter library to selection at different time points, up to 6–7 days, within the macrophages. We appreciate that the present investigation is not a comprehensive study because the promoter library may not be complete as random restriction by Sau3AI has most certainly led to the loss of some promoter fragments involved in intracellular growth. Fragmenting chromosomal DNA by other means may thus be helpful to construct more representative libraries to be used in the context of this vector for the identification of promoters up- or down-regulated under specific environmental conditions.
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ACKNOWLEDGEMENTS |
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Edited by: W. Bitter
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 26 July 2006;
revised 7 November 2006;
accepted 9 November 2006.
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