Unité de Génétique Mycobactérienne, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France1
Author for correspondence: Brigitte Gicquel. Tel: +33 1 45 68 88 28. Fax: +33 1 45 68 88 43. e-mail: bgicquel{at}pasteur.fr
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ABSTRACT |
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Keywords: Mycobacterium tuberculosis, macrophage, gene induction, fluorescence
Abbreviations: GFP, green fluorescent protein; (r)BCG, (recombinant) bacille CalmetteGuérin; FACS, fluorescence-activated cell sorting
a Present address: SmithKline Beecham Biologicals, Laboratory of Molecular Bacteriology, Rue de lInstitut 89, B-1330 Rixensart, Belgium.
b Present address: INSERM U411, Faculté de Médecine Necker-Enfants Malades, 156, Rue de Vaugirard, 75730 Paris Cedex 15, France.
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INTRODUCTION |
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The results of previous studies with other intracellular bacteria, such as Salmonella and Listeria, indicate that the adaptation to an intracellular environment involves modification in the pattern of bacterial gene expression (Moors & Portnoy, 1995 ). By analogy, one would assume that similar mechanisms are employed by M. tuberculosis and to this end the identification of those M. tuberculosis genes whose expression is preferentially up-regulated within host cells would contribute significantly to our understanding of the organisms pathogenicity. The application of the Aequoria victoria green fluorescent protein (GFP) as a selectable marker to analyse bacterial gene expression has enhanced the development of methods to identify in vivo induced genes, such as the strategy termed differential fluorescence induction (DFI) (Valdivia & Falkow, 1996
). This technique permits the assessment of promoter activity in different environmental conditions by the analysis of GFP fluorescence, with recombinant bacteria of interest capable of being separated by fluorescence-activated cell sorting (FACS). This system was initially applied to the isolation of pH-regulated promoters of Salmonella typhimurium (Valdivia & Falkow, 1996
) and later extended to identify promoters of the same organism showing differential activity outside and within host cells (Valdivia & Falkow, 1997
). The observation that GFP is efficiently expressed in mycobacteria and that fluorescent bacteria can be directly observed in infected macrophages (Dhandayuthapani et al., 1995
; Kremer et al., 1995
) suggested that such a strategy may be applicable to the study of gene induction by mycobacteria in host cells. A similar system to DFI was recently used to identify promoters of Mycobacterium marinum which show elevated levels of expression within macrophages compared to the extracellular environment (Barker et al., 1998
).
While GFP represents a useful tool for the study of mycobacterial genetics, another genetic system proving extremely advantageous is that based on the sacB gene of Bacillus subtilis. This gene encodes a secreted enzyme that confers sucrose sensitivity on mycobacteria (Pelicic et al., 1996a ). This property has been used to efficiently perform allelic exchange and construct transposon mutant libraries in both the fast- and slow-growing mycobacteria (Pelicic et al., 1996b
, 1997
). In this study, we have combined the features of both the GFP and SacB proteins to develop a system permitting the isolation of genes of M. tuberculosis differentially expressed within host cells. Macrophages were infected with recombinant (r) Mycobacterium bovis bacille CalmetteGuérin (BCG) containing a library of M. tuberculosis promoters controlling gfp and sacB expression, and GFP-positive bacteria sorted by FACS. SacB expression was then used to eliminate rBCG clones with strong promoter activity outside the macrophage, leading to the enrichment of clones containing M. tuberculosis promoters differentially expressed intracellularly.
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METHODS |
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Vector construction.
The promoterless B. subtilis sacB gene was amplified from the pJQ200 vector (Quandt & Hynes, 1993 ) using the primers SacB1 (5'-GGGGCTGCAGAAAAAGGAGACATGAACGATGAACATC) and SacB2 (5'-GGGGCTGCAGTATGGGATTCACCTTTATGTTGATAAG) . The PstI-digested product was cloned into PstI-digested pKEN-GFPmut2 (Cormack et al., 1996
), placing the sacB gene directly downstream of a promoterless, highly fluorescent version of the Aequoria victoria gfp gene to give plasmid pIPX39. We next constructed a suitable mycobacterialE. coli shuttle vector for subsequent cloning procedures. The kanamycin resistance gene was cloned into NdeI/BsaI-digested pUC18, resulting in plasmid pPV8. The origin of replication of the M. fortuitum plasmid pAL5000 (Ranes et al., 1990
) was then cloned into the StuI site of pPV8. The resulting plasmid, pPV24, is a multipurpose shuttle cloning vector harbouring five unique restriction sites (KpnI, BamHI, XbaI, PstI, SphI) and allows blue/white selection of recombinants in E. coli. The plasmid pJFX1 was constructed by digesting pIPX39 with BamHI and SphI and cloning of the resulting 2·2 kb fragment into BamHI/SphI-digested pPV24. The tT4 transcription terminator was amplified from the vector pJEM12 with primers TT4.FOR (5'-AGACTAGATCTGTCATGCCATCCG) and TT4.REV (5'-TATCCGGATCCGCGGGCCCAG), digested with BamHI and BglII, and cloned into BamHI-digested pJFX1, giving pJFX2. The strong ß-lactamase promoter of Mycobacterium fortuitum (Timm et al., 1994
) was cloned into ScaI/BamHI-digested pJFX2 to give pJFX4.
Construction of the M. tuberculosis genome library.
M. tuberculosis 103 DNA was extracted as previously described (Otal et al., 1991 ) and digested with Sau3A (0·03 U per 5 µg) for 20 min. DNA was run on low-melting-temperature agarose (Bio-Rad), then the 200 bp1·5 kb fraction was purified (QIAquick gel extraction kit, Qiagen) and ligated to dephosphorylated, BamHI-digested pJFX2. DNA was extracted from approximately 10000 recombinant colonies and electroporated into M. bovis BCG.
Macrophage preparation and infection.
Bone-marrow-derived macrophages were isolated from the femurs of 7-week-old female Balb/c mice. Cells were seeded at 2 x 105 macrophages per well in 24-well plates (Costar) and incubated at 37 °C in 5% CO2 for 7 d. Macrophage monolayers were infected with bacteria at a m.o.i. of 1:1. After 4 h of infection, extracellular bacteria were removed by washing four times with PBS and incubation continued at 37 °C in 5% CO2.
FACS analysis and sorting of fluorescent macrophages.
Bacteria and macrophages were analysed with a FACScan (BectonDickinson) and sorted with an ELITE cell sorting system (Beckman Coulter Inc.). After 56 d of infection, BCG-infected macrophages were washed three times in PBS, scraped into 1 ml PBS and analysed by FACS. Macrophages exhibiting fluorescence levels greater than those infected with M. bovis BCG harbouring pJFX2 alone were collected. To recover bacteria, sorted macrophages were centrifuged and lysed in water plus 0·1% Tween 80. Recovered bacteria were grown in 7H9 medium for 7 d and macrophage infection and sorting repeated until the desired level of enrichment of macrophage/fluorescent bacteria was attained.
Selection of clones with enhanced intracellular expression.
Macrophages in 24-well culture plates were infected with recombinant BCG clones recovered after the selection process at a m.o.i. of 1:1. After 4 h of infection, macrophages were washed four times with PBS and the infection continued or bacteria recovered from Tween-80-lysed macrophages for FACS determination of fluorescence levels (representing day 0). After 6 d of infection, the remaining macrophages were washed four times with PBS, lysed and bacterial fluorescence levels determined by FACS. Clones displaying a day 6 to day 0 fluorescence ratio of approximately 2 or greater were considered to display significant intracellular induction. A total of 2000 bacteria from culture medium alone or lysed macrophage were analysed as a function of side scatter and GFP fluorescence. Quantification of fluorescence levels measured by FACS was determined by the use of the Lysis II program (BectonDickinson), while fluorescence of bacterial suspensions and colonies was visualized directly using a Axioscop fluorescence microscope (Zeiss).
PCR and sequencing of selected clones.
DNA from clones of interest was extracted by freeze-thawing of bacterial suspensions and subjected to PCR amplification using primers BCGPR.FOR1 (5'-AACGGGATCCAAGCATAAAGCTAGT) and BCGPR.REV (5'-TTACGGATCCGTATATCTCCTTCTT). PCR fragments were sequenced using a model 373 automated DNA sequencer (Applied Biosystems). DNA homologies were determined using the M. tuberculosis BLAST server at the Sanger Centre (www.sanger.ac.uk).
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RESULTS |
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The growth of M. bovis BCG harbouring pJFX2 on plates containing 0·5 and 5% sucrose was not affected, while M. bovis BCG carrying pJFX4 was killed in the presence of 0·5 and 5% sucrose (data not shown). This suggested that expression of the sacB gene of pJFX2 was sufficient to permit effective counterselection of rBCG. We next determined if such sacB expression could permit the regulated elimination of sacB-expressing bacteria. A promoter library of M. tuberculosis 103 DNA (0·21·5 kb) was constructed in pJFX2 and transformed into M. bovis BCG, resulting in a pool of BCG clones exhibiting varying levels of GFP expression as assessed by FACS (see Fig. 3a).To obtain a population containing a sufficient proportion of gfp/sacB-expressing bacteria to allow detailed analysis, GFP-positive M. bovis BCG from the pool of clones were sorted by FACS and the subsequent population re-analysed. After one round of sorting, the proportion of GFP-positive BCG rose from 9 to 41%, with varying levels of fluorescence (Fig. 2a
). As sacB and gfp are present in an operon, then it can be assumed that a similar variety in sacB expression is present in this library. To test and monitor the regulation of counterselection by the use of sucrose, the effect of both sucrose concentration and incubation time on the killing of sacB-expressing bacteria was analysed. The pooled BCG clones were grown to an OD600 of 0·25 at which point sucrose, ranging in concentration from 0·01 to 3%, was added and the proportion of GFP-positive bacteria analysed by FACS at varying time points. As seen in Fig. 2(b)
, while no or low concentrations of sucrose (0·1%) had a minimal effect on the proportion of GFP-positive bacteria observed, increasing the concentration of sucrose in the medium led to an increased elimination of GFP-positive bacteria, in a dose-dependent manner. Analysis of individual FACS profiles (Fig. 2a
) suggested that it was those bacteria expressing high levels of gfp/sacB that were being preferentially eliminated by the effect of sucrose. To confirm this, we determined the mean fluorescence level of GFP-positive bacteria over time after the addition of 0·3% sucrose to the M. bovis BCG pool. As shown in Fig. 2(c)
, the mean fluorescence of the population steadily decreased with time, indicating that it was strong gfp/sacB-expressing bacteria that were being eliminated initially, with longer periods of incubation required to eliminate bacteria expressing intermediate or low levels of gfp/sacB. Taken together, these results suggest that varying the concentration of sucrose and time of incubation can be used to achieve selective elimination of sacB-expressing bacteria based on the strength of the promoter that they harbour.
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Colonies obtained after growth on solid medium were cultured individually in 24-wells plates and used to infect macrophages for 6 d. The level of fluorescence in 7H9 medium and in macrophages 4 h or 6 d after infection was compared by FACS. We considered detection of fluorescence in macrophages just after infection (4 h) to be a good measure of initial levels of gfp expression as it accounts for any effect on fluorescence due to exposure to the culture medium and/or the preparation of bacteria from macrophages. As such, we calculated the level of intracellular induction as the GFP level at day 6 of infection compared to 4 h (termed day 0). Typically, the fluorescence level of clones grown in 7H9 culture medium was either the same or slightly lower than that observed for bacteria recovered from macrophages after the 4 h infection period (data not shown). Out of the approximately 400 individual rBCG clones examined, seven showed reproducibly greater levels of fluorescence after 6 d within the macrophage compared to GFP fluorescence at day 0 (Table 1). Three of these clones (13) were isolated from bacteria that did not undergo sucrose selection, while the remaining four (clones 47) were isolated by the combined fluorescence induction and sucrose counterselection procedures. A representative histogram of one of the isolated clones is shown in Fig. 3(d)
. Calculation of the increase in fluorescence demonstrated a range in intracellular induction of approximately two to threefold (Table 1
).
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Of the seven clones, four showed strong similarity to previously described genes and coding sequences from a number of species (Table 1). Clone 1 corresponds to a gene exhibiting strong homology to transketolases of the pentose phosphate pathway. Clone 2 maps to the fadB4 gene, homologous to a 3-hydroxyl-CoA dehydrogenase, postulated to be involved in fatty acid metabolism. This gene is also strongly similar to quinone oxidoreductases, which serve a role as phase II detoxifying enzymes required for defence against the intake of xenobiotics. Clones 4 and 5 both correspond to genes possibly involved in the metabolism of sulphur. The sseA gene (clone 4) encodes a thiosulfate sulfurtransferase, which catalyses sulfur transfer from thiosulfate. The cysD gene (clone 5) encodes a sulfate adenylate transferase subunit B, which is required for the activation of inorganic sulphate. Immediately downstream of cysD lies cysN which encodes the A subunit of the sulfate adenylate transferase complex. Clone 6 contains the postulated promoter region of a gene encoding an alanine/glycine-rich protein which is a member of the large PE-PGRS subfamily of M. tuberculosis (Cole et al., 1998
).
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DISCUSSION |
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The levels of intracellular induction obtained for the isolated promoters was reproducibly in the range of two to threefold. As GFP requires no substrate and thus no amplification step for the visualization of fluorescence, one could consider that an induction of threefold represents an actual threefold increase in expression, which would constitute a significant change in protein levels. Accordingly, most studies examining gene regulation from other mycobacteria have shown a similar level of induction. For example, 11 of 12 M. marinum promoters shown to be induced in vivo exhibited GFP induction levels of 1·43·4-fold; the one exception was a gene induced up to 15-fold (Barker et al., 1998 ). In contrast to these results obtained with mycobacteria, studies with Salmonella using a similar mutant form of GFP showed in vivo induction typically in the range of 830-fold, with one promoter induced up to 443-fold (Valdivia & Falkow, 1997
). This difference may suggest a refining of host-cell-associated gene expression by M. tuberculosis such that small changes in expression are sufficient to exert the desired biological action, indicating an exquisite adaptation of the bacteria to its host. It is also possible that M. tuberculosis promoters strongly induced in vivo are present but were not identified in this study; for example, the M. tuberculosis acr gene appears to be induced around 100-fold within the murine macrophage, although this level was determined using the luciferase reporter gene and thus cannot accurately be compared to results obtained in this study using the GFP (Yuan et al., 1998
).
This study selects for promoters that increase their level of expression within the macrophage and sustain such enhanced expression over a prolonged period of time (i.e. at least 6 d). Thus we cannot exclude the fact that promoters switched on early and then down-regulated may have been eliminated by the use of our criteria. The GFPSacB system could be modified accordingly to select for promoters with transient expression by changing the time of infection at which bacteria are analysed. It is also possible that by using a macrophage infection time of 4 h as our zero time point we could underestimate the level of induction if a promoter has already become active during this 4 h period. This does not seem to be the case for the promoters identified in this study, as levels of fluorescence in media and after infection for 4 h were essentially the same.
Our strategy to isolate promoters active in vivo made use of a novel system, where the properties of the A. victoria GFP and B. subtilis SacB protein were combined to enrich for the promoters of interest. Previously, the SacB protein has been shown to be an extremely powerful tool in the field of mycobacterial genetics. The ability to specifically eliminate sacB-expressing mycobacteria in the presence of sucrose has been exploited to allow efficient allelic exchange and construction of transposon mutant libraries in both the slow- and fast-growing mycobacteria (Pelicic et al., 1997 ). This is of particular importance in M. tuberculosis, where these techniques were always considered technically difficult and relatively inefficient. In all these studies, constitutive sacB expression was used at single sucrose concentrations to perform the counterselection. Our results demonstrate another use of this counterselective property of SacB, where recombinant bacteria could be selectively eliminated depending on their level of sacB expression. Such counterselection could be finely regulated depending on the concentration of sucrose used and the time of incubation. The demonstration that the level of sacB expression by mycobacteria corresponds to the level of killing may be exploited to study other facets of mycobacterial gene regulation; for example, this property was used to successfully isolate regulatory genes of Rhodobacter sphaeroides (Sabaty & Kaplan, 1996
).
Mycobacterial genes that undergo specific up-regulation within host cells may be important for the intracellular survival and virulence of the bacterium. Thus the deletion of such genes could provide M. tuberculosis strains limited in virulence, which may form the basis of attenuated vaccines to combat tuberculosis.
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ACKNOWLEDGEMENTS |
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Received 28 April 1999;
revised 21 June 1999;
accepted 25 June 1999.