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Appl Environ Microbiol. 2012 October; 78(19): 6829–6837.
PMCID: PMC3457510

Mycobacterial Shuttle Vectors Designed for High-Level Protein Expression in Infected Macrophages


Mycobacterial shuttle vectors contain dual origins of replication for growth in both Escherichia coli and mycobacteria. One such vector, pSUM36, was re-engineered for high-level protein expression in diverse bacterial species. The modified vector (pSUM-kan-MCS2) enabled green fluorescent protein expression in E. coli, Mycobacterium smegmatis, and M. avium at levels up to 50-fold higher than that detected with the parental vector, which was originally developed with a lacZα promoter. This high-level fluorescent protein expression allowed easy visualization of M. smegmatis and M. avium in infected macrophages. The M. tuberculosis gene esat-6 was cloned in place of the green fluorescence protein gene (gfp) to determine the impact of ESAT-6 on the innate inflammatory response. The modified vector (pSUM-kan-MCS2) yielded high levels of ESAT-6 expression in M. smegmatis. The ability of ESAT-6 to suppress innate inflammatory pathways was assayed with a novel macrophage reporter cell line, designed with an interleukin-6 (IL-6) promoter-driven GFP cassette. This stable cell line fluoresces in response to diverse mycobacterial strains and stimuli, such as lipopolysaccharide. M. smegmatis clones expressing high levels of ESAT-6 failed to attenuate IL-6-driven GFP expression. Pure ESAT-6, produced in E. coli, was insufficient to suppress a strong inflammatory response elicited by M. smegmatis or lipopolysaccharide, with ESAT-6 itself directly activating the IL-6 pathway. In summary, a pSUM-protein expression vector and a mammalian IL-6 reporter cell line provide new tools for understanding the pathogenic mechanisms deployed by various mycobacterial species.


Mycobacterium tuberculosis has killed millions of people since the beginning of recorded human history (9, 14, 15). Every year, 12 to 14 million people suffer from active disease, and of these 2 million die. For those apparently cured from such syndromes as tuberculous (TB) meningitis, more than 60% are likely to die within 5 years, and close to half of those who survive will develop permanent medical problems (32, 38). While therapy for TB has been lifesaving, preventing infections and disease progression would be much more effective. M. avium and M. leprae are two additional mycobacterial species of concern for the human population, with immunocompromised patients being very susceptible to M. avium. There are no broadly effective vaccines for mycobacterial infections. An attenuated derivative of M. bovis (the bacillus Calmette-Guérin strain, or BCG) is the most commonly used vaccine for TB. However, BCG has limited effectiveness in newborns and children, is not protective in adults, and thus is not currently recommended in the United States (3). Indeed, BCG does not prevent the reactivation of latent TB to clinical disease. In a mouse study, BCG vaccination enhanced the virulence of the most menacing of M. tuberculosis genotypes in terms of drug resistance and transmission, the Beijing strain (26). In a separate guinea pig model, multiple sequential vaccinations with BCG reduced survival following subsequent challenges with virulent M. tuberculosis (5). With 11 new recombinant vaccines, all but one depends on boosting with BCG (1, 8, 23). A promising alternative candidate has emerged with a recombinant M. smegmatis (43). Even with such advances, it is imperative that better tools to study the pathogenic mechanisms of M. tuberculosis be developed, especially because M. tuberculosis has a large, complex genome comprised of ~4.4 million base pairs with approximately 4,000 open reading frames (12). About 1,500 M. tuberculosis genes have no known functions.

During infection, M. tuberculosis can exhibit both an active phase and a long-term, poorly understood latency that can last several decades in infected individuals. The latency likely involves the suppression of innate inflammatory pathways that are more defined early in infection. For example, avirulent strains of mycobacteria induce higher levels of inflammatory cytokines (tumor necrosis factor [TNF], nitric oxide, and interleukin-6 [IL-6]) in infected macrophages than do pathogenic strains (7). This is partly due to several M. tuberculosis genes that inhibit innate inflammatory pathways (12, 42). The best understood involves the ESX-1 type VII secretion system, a protein transport system that releases multiple proteins across the cell envelope (20, 21, 34, 42). One substrate of this pathway is EspR, a secreted transcription factor that controls M. tuberculosis virulence, partly by functioning in a negative feedback loop (34). Two additional substrates include early-secreted antigenic target protein 6 (ESAT-6) and CFP-10, which exist in a 1:1 heterodimer complex (21, 36). ESAT-6 attenuates Toll-like receptor signaling pathways, resulting in diminished production of inflammatory cytokines, including IL-12, IL-6, and TNF (33). The inhibition occurs following a direct binding of ESAT-6 to Toll-like receptor 2 (TLR2), which subsequently antagonizes TLR2-, TLR4-, and TLR9-mediated release of inflammatory cytokines (33). However, ESAT-6 has also been reported to enhance cytokine production (11). Moreover, ESAT-6 has additional proposed functions, including a lysin and an inflammasome activator (10, 17, 22, 30). Using M. marinum infections in zebra fish larvae, ESAT-6 enhances mycobacterial spread early in infections by elevating matrix metalloproteinase 9 (MMP9) expression in epithelial tissues, which functions as a macrophage chemoattractant (16, 42, 44). Thus, while it is clear that ESAT-6 functions in early infection stages when M. tuberculosis is actively replicating, its mechanism of action remains complicated.

The objectives of the current study were to develop mycobacterial vectors that provide high-level protein expression and develop novel mammalian reporter cell lines for studying mycobacterial infections.


Bacterial strains, plasmids, and antibodies.

The parental pSUM plasmids were originally developed and described elsewhere (2). pSUM36 was used in the current studies, with transformations done into chemically competent E. coli (XL1-Blue or HB101) or electrocompetent M. smegmatis or M. avium (subsp. homimissuis) (ATCC 700084 and 700898, respectively; American Type Culture Collection, Manassas, VA). E. coli cells were grown in Luria-Bertani (LB) broth (Miller's LB broth; Thermo-Fisher) or plated on LB agar. M. smegmatis and M. avium cells were grown in Difco Middlebrook 7H9 broth supplemented with oleic acid-albumin-dextrose-catalase (OADC) and Tween 80 at a 0.5% (vol/vol) final concentration. The cells were plated on Middlebrook 7H10 agar plates supplemented with 10% OADC. Kanamycin was added at a final concentration of 10 and 50 μg/ml for M. smegmatis and M. avium, respectively. Genomic DNA from M. tuberculosis H37Rv was obtained from Colorado State University under a TB Vaccine Testing and Research Materials Contract with the National Institutes of Health (N01-AI-40091). The plasmid pMRLB7, obtained under the same contract, contains histidine (His)-tagged ESAT-6. The green fluorescent protein (GFP) gene was originally modified for expression in prokaryotes, and this version included an optimized translational initiation region from the plasmid pGreenTIR as described earlier (29). The GFP gene was cloned from pGreenTIR into the HindIII site of the pSUM36 vector (pSUM-lacZ-gfp) (2). The groEL promoter was amplified from M. tuberculosis genomic DNA using standard PCRs. A BamHI restriction site was included at the 5′ end. This fragment was cloned into the pCR2.1 TOPO-TA cloning vector and subsequently isolated as a BamHI/XbaI piece that was subcloned into pSUM36 cut with BamHI and NheI to remove the lacZα promoter. The GFP gene was subcloned into the HindIII site of this new pSUM-groEL vector. The HindIII fragment containing gfp was also cloned in the reverse orientation in pSUM36, resulting in it being expressed off the 3′ end of the kanamycin cassette (pSUM-kan-MCS1-gfp). This vector was subsequently modified to reorient the BamHI and HindIII restriction sites to facilitate directional cloning and introduce the Shine-Dalgarno sequence in the correct orientation (pSUM-kan-MCS2). esat-6, without a His tag, was subcloned into the HindIII site of this new vector, enabling expression of ESAT-6 to be driven by the kanamycin cassette (pSUM-kan-MCS2-esat-6). The various pSUM plasmids were electroporated into chemically competent E. coli or electrocompetent M. smegmatis and M. avium. The resulting clones were monitored for GFP expression by flow cytometry on a FACSCalibur (Becton, Dickinson, San Jose, CA). Data were analyzed using FloJo software (Tree Star, Inc., Ashland, OR). When using M. avium cultures, both the mycobacterial and mammalian cells were fixed with 4% paraformaldehyde prior to imaging studies. Bacterial cell extracts were prepared by bead beating, and the protein component was precipitated with the addition of 10% (vol/vol; final concentration) trichloroacetic acid as detailed elsewhere (13). Total protein or immune-precipitated proteins were resolved on low-molecular-mass protein separation gels as described previously (37). A monoclonal antibody directed against ESAT-6 was used for Western immunoblotting experiments according to the manufacturers' instructions (HYB 076-08; Abcam, Cambridge, MA). In certain experiments, ESAT-6 was immunoprecipitated from 50 ml of M. smegmatis culture supernatants. After 3 days of culture, the mycobacteria were pelleted by centrifugation and the supernatant sterilized by filtration (0.22-μm filter). The filtered supernatant was mixed with 50 μl of protein A gel slurry in the presence of 6 μl of anti-ESAT-6 polyclonal antisera (equal mix of antisera from Pierce [catalog no. PA1-19446] and Cedarlane [catalog no. CLX305AP]; Thermo-Fisher, Inc.; Cedarlane Labs, Burlington, NC). After 4 to 5 h of mixing, the antibody (Ab)-protein A beads were centrifuged at 2,000 rpm for 10 min, followed by 4 washes in 20 mM Tris-Cl, pH 7.60, containing 0.15 M NaCl and 1% Triton X-100. The remaining pellet was boiled in SDS sample buffer and the material resolved on low-molecular-weight gels.

Recombinant ESAT-6 purification.

ESAT-6(His6) was purified as described previously (40), with the following modifications. Following sonication, the cleared lysate was applied to a HiTrap immobilized metal affinity column (GE Biosciences). Prior to elution in 1 M imidazole, the column was washed with 0.5% 3-[N,N-dimethyl(3-myristoylaminopropyl) ammonio] propanesulfonate amidosulfobetaine-14 (ASB-14) and reconstituted in 10 mM Tris-Cl, pH 7.9. This zwitterionic detergent is used to remove contaminating lipopolysaccharide.

Macrophage reporter cell lines.

The J774A.1 murine macrophage cell line was obtained from the American Type Culture Collection. These cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum, 5 × 10−5 M 2-mercaptoethanol, penicillin (100 U/ml final concentration), streptomycin (100 μg/ml final concentration), and 10 mM glutathione. The macrophages were plated at 3 × 105 cells/ml 1 day prior to infection. M. smegmatis or M. avium clones containing the lacZα-, groEL-, and kan-driven GFP plasmids, as well as the parental vector, pSUM36, were used to infect the J774A.1 macrophage cells at various multiplicities of infection (MOI). After 3 h of infection, the cells were washed with DMEM containing 10% (vol/vol) fetal calf serum and 0.05% (vol/vol) Tween 80. Cells were incubated with amikacin for 45 min to limit the extracellular growth of the mycobacteria. GFP expression was monitored at 24 h postinfection by fluorescence microscopy. The murine Il-6 promoter was amplified from genomic DNA (extracted from tail biopsy specimens from C57BL/6 mice) with standard PCRs (4). AseI and AgeI sites were included at the 5′ and 3′ ends, respectively. The resulting fragment was subcloned into an AseI/AgeI-digested pEGFP-N1 plasmid (Clontech, BD Biosciences, Inc.). The new pmIl-6-gfp plasmid was electroporated into J774A.1 macrophages. Individual clones were isolated by G418 selection, with one clone (J774A.1-Il-6-gfp #3) being used for the studies described here. This clone was maintained in the aforementioned DMEM supplemented with 1 mg/ml of G418. These cells were infected with M. smegmatis at MOIs of 30:1, 10:1, and 3:1. IL-6 activation was determined 24 h postinfection by fluorescence microscopy. Alternatively, IL-6 activation was monitored by GFP expression using flow cytometry on a FACSCalibur instrument.

Microscopy imaging.

A Nikon Eclipse T5100 microscope was used for light and fluorescence microscopy. Confocal microscopy was done with a pDV Deltavision deconvolution microscope equipped with a 100× (1.4 numeric aperture; APO) Olympus lens in Cool Snap HQ2 camera with fluorescein isothiocyanate (FITC)/CY5 filter sets for all fluorescence microscopy experiments. A single differential interference contrast (DIC) reference image and fluorescent z-stacks (0.2-μm z-step) were taken for each cell. Images were background subtracted, and fluorescent channel overlays were performed in ImageJ ( Representative fluorescent z-plane images and movies of the entire z-volume of each cell are provided in the supplemental material.

Statistical analyses.

For flow cytometry comparisons between different bacterial clones and strains, an unpaired Student's t test was used for determining the significance. Statistical significance was indicated by one (*, P < 0.005), two (**, P < 0.0005), or three asterisks (***, P < 0.0001).


Development of high-level protein expression shuttle vectors.

The pSUM plasmids are a family of shuttle vectors designed for replication in both E. coli and mycobacteria (2). The vectors contain multiple cloning sites, lacZα for recombinant screening, and a kanamycin resistance gene, kan. These vectors have been useful for screening genomic libraries (35). We wanted to determine whether such vectors were suitable for high-level protein expression in both E. coli and distinct mycobacterial strains. The gene encoding a modified GFP, under the control of an optimized translational initiation region, was subcloned into the parental pSUM36 vector under the control of the lacZα promoter (pSUM-lacZ-gfp) (Fig. 1A) (29). To determine whether different promoter cassettes could increase GFP expression, the groEL promoter, cloned from M. tuberculosis, was substituted for lacZα (pSUM-groEL-gfp). A third expression vector was developed in which the kanamycin cassette, already part of the original pSUM vector, was used to express GFP (pSUM-kan-MCS1-gfp). The parental plasmid, pSUM36, and each of the gfp expression vectors were transformed into E. coli. Transformants were grown overnight, and the levels of GFP expressed by each were determined by flow cytometry. E. coli cells containing plasmids with the lacZ-(pSUM-lacZ), groEL-(pSUM-groEL), and kan-(pSUM-kan-MCS1) promoters had statistically significant 3-, 3-, and 9-fold increases in fluorescence, respectively (mean fluorescence intensity [MFI]), compared to control plasmid transformants (P < 0.0005) (Fig. 1B). The overexpression of GFP at different levels had no effect on E. coli growth, as similar CFU/ml were obtained with control and high-GFP-expressing clones (Fig. 1C). The same plasmids were subsequently transformed into M. smegmatis, and the fluorescence intensity of the mycobacterial clones was compared after 3 days of culture (Fig. 1D). There was a 7-, 9-, and 190-fold increase in the MFI, respectively (P < 0.0005) (Fig. 1D). There was no impact of these high-level protein expression cassettes on the mycobacterial growth characteristics (Fig. 1E). These results demonstrate that the kanamycin promoter/gene cassette was the most effective for GFP expression in both E. coli and M. smegmatis, with the levels detected in the mycobacterial species being 21-fold higher than those of standard E. coli strains (Fig. 1B and andD).D). Given the success of the kanamycin cassette, the parental pSUM36 vector was redesigned to facilitate directional cloning. The HindIII and BamHI restriction sites were flipped, and the optimized translation initiation site was cloned at the 3′ end of the kanamycin cassette (pSUM-kan-MCS2). While this new cloning vector resulted in GFP expression in E. coli equivalent to that with the pSUM-kan-MCS1 plasmid, it provided a 2-fold higher level of fluorescence in M. smegmatis than that achieved with pSUM-kan-MCS1 (Fig. 1B and andD).D). The experiments were repeated in M. avium. A 500-fold increase in fluorescence was detected over the control vector-transformed M. avium clones, with limited impact on mycobacterial growth (Fig. 1F and andGG).

Fig 1
Mycobacterial shuttle vectors with distinct promoter cassettes provide high-level protein expression. (A) The original pSUM36 vector and its modified derivatives are shown. In the parental vector, gfp was cloned downstream of the lacZα promoter ...

GFP-expressing mycobacteria are detected in infected macrophages.

Previous studies have shown that mycobacterial strains expressing GFP can be visualized in infected macrophages (18, 25). To determine whether the pSUM-modified vectors were suitable for detecting mycobacteria in cell lines, macrophages were infected at an MOI of 30:1 and the fluorescence levels assayed 24 h later with fluorescence microscopy. M. smegmatis containing the parental vector could not be detected in the infected cells (Fig. 2A). The mycobacterial subclones expressing GFP from the lacZα and groEL promoters were evident as small, faint green bacilli in the macrophages (Fig. 2A). M. smegmatis clones expressing GFP under the control of the kanamycin cassette (pSUM-kan-MCS1-gfp) yielded highly visible mycobacteria within the macrophages. The pSUM-kan-MCS2-gfp construct provided the most fluorescently intense mycobacteria. To determine whether the different levels of GFP expression affected virulence and host cell survival, the cells were incubated with a LIVE/DEAD blue fluorescent reactive dye, a dye that permeates compromised cellular membranes, resulting in an intense fluorescent stain. Macrophages cultured in the presence of 0.1% NaN3 exhibited an intense blue fluorescence under UV light, consistent with the loss of membrane integrity. In contrast, the macrophages exhibited a normal viability, even after infection with the different mycobacterial clones, independent of GFP expression (Fig. 2A, middle). Bright- field images revealed evenly distributed macrophage monolayers that were unaffected by the type of expression vector used (Fig. 2A, lower). These experiments indicated that the pSUM vectors with the kanamycin cassette coupled to an optimized translational initiation site were ideal for achieving high-level protein expression in infected cells. To determine if these vectors were suitable for other mycobacterial species, M. avium was transfected with pSUM-kan-MCS2-gfp. The resulting clones were used to infect macrophages at MOIs of 3:1, 10:1, and 30:1 (Fig. 2B). As with M. smegmatis, the M. avium-GFP clones were highly visible in the infected macrophages (Fig. 2B, upper). The infected macrophages exhibited similar viabilities at the different MOIs, as determined by LIVE/DEAD staining and bright-field images (Fig. 2B, middle and lower). M. marinum clones expressing GFP have a reduced virulence in zebra fish infection models (31). To determine if the M. smegmatis GFP-overexpressing clones were attenuated in the macrophages, the CFU/ml were calculated after lysing the infected macrophages. While there were variations in the CFU/ml calculated for the different M. smegmatis clones, we did not detect statistically significant differences between the clones expressing low and high levels of GFP (Fig. 2C). Given our ability to achieve high-level GFP expression in mycobacteria, we next assessed whether these would be suitable for detecting the location of mycobacteria in infected macrophages. Confocal microscopy analyses of the infected macrophages revealed distinct mycobacterial morphologies between M. smegmatis and M. avium. M. smegmatis cells were seen as extended filaments that overlapped with the phagolysosome, as detected by LAMP staining. This pattern was distinct from the M. avium cells, which appeared clustered in discrete, rounded foci that again were in proximity to lysosomal granules (LAMP) (Fig. 3A versus versusB).B). The differences between M. avium and M. smegmatis were clearly revealed with multiple z-plane stacks (see the movies in the supplemental material).

Fig 2
M. smegmatis and M. avium clones expressing high levels of GFP are easily visualized in infected macrophages. (A) M. smegmatis transformants containing different GFP expression plasmids were used to infect macrophages at a multiplicity of infection (MOI) ...
Fig 3
M. smegmatis and M. avium clones expressing high levels of GFP exhibit distinct morphologies in infected macrophages. (A) M. smegmatis transformants containing different GFP expression plasmids were used to infect macrophages at a multiplicity of infection ...

High-level ESAT-6 expression is insufficient to suppress innate inflammatory pathways in macrophages.

To further examine the usefulness of the modified pSUM vectors for studying high-level protein expression, the gfp gene was replaced with esat-6 (Fig. 1A). ESAT-6 is normally secreted via the ESX-1 secretion system and is proposed to inhibit Toll-like receptor signaling (33, 34, 42). To compare the expression levels of ESAT-6, extracts were prepared from E. coli and M. smegmatis clones containing either the parental vector or the pSUM-kan-MCS2-esat-6 expression vector (Fig. 4A). M. smegmatis containing esat-6 was grown in 7H9 medium under culture conditions that prevent ESAT-6 secretion that would enable us to detect the protein in mycobacterial lysates (13). Western immunoblotting confirmed that ESAT-6 was specifically expressed at high levels in the M. smegmatis clones containing the enhanced protein expression plasmid (Fig. 4A, lanes 6 and 7). ESAT-6 was almost undetectable in the E. coli extracts, consistent with the lower levels of GFP expression in E. coli versus the mycobacterial strains (Fig. 4A, lanes 3 and 4). Even though M. smegmatis was grown in medium that does not allow ESAT-6 secretion, it was unknown whether ESAT-6 would be released with the high-level protein expression vector (13). To determine whether ESAT-6 was released into the medium, M. smegmatis cultures were harvested after 3 days of culture. A polyclonal antisera was used to precipitate ESAT-6 from the clarified and filtered culture supernatant. This polyclonal antisera precipitated an exogenous source of pure ESAT-6, produced in E. coli, that was added directly into 1.0 and 50 ml of lysis buffer, as determined by subsequent Western immunoblotting (Fig. 4B, lanes 1 to 2). In contrast, no endogenously produced ESAT-6 was precipitated from the clarified M. smegmatis culture supernatants (Fig. 4B, lane 3). To confirm that the culture supernatant did not contain a protease activity that might have degraded ESAT-6, a separate immunoprecipitation was performed in which pure ESAT-6 was spiked into the culture supernatant. This exogenously added ESAT-6 was detected in the precipitate (Fig. 4B, lane 4). Taken together, these findings indicate that the modified pSUM vectors provided for high-level protein expression in diverse mycobacterial species.

Fig 4
Mammalian IL-6 reporter cell line responsive to innate inflammatory stimuli. (A) ESAT-6 protein expression in E. coli (lanes 2 to 4) and M. smegmatis (lanes 5 to 7) transformed with pSUM-empty vector (lanes 2 and 5) or pSUM-kan-MCS2-esat-6 (lanes 3, 4, ...

Development of an IL-6-driven GFP macrophage reporter cell line responsive to inflammatory stimuli.

Macrophages secrete inflammatory cytokines, such as IL-1, IL-6, IL-12, and TNF-α, in response to bacterial infections and/or bacterial products such as lipopolysaccharide (LPS). To monitor innate cell activation, a murine Il-6 promoter (600 bp) was cloned upstream of GFP, replacing the standard cytomegalovirus promoter (Fig. 4C) (4). This construct was used to generate stable macrophage cell lines (J774A.1) expressing GFP under the control of the IL-6 promoter. One subclone (J774A.1-Il-6-gfp #3) exhibited a >7-fold increase in GFP expression after the cells were cultured for 24 h in the presence of LPS. A dose-response curve revealed that the cells were sensitive to LPS concentrations as low as 1 ng/ml (data not shown). These J774A.1 Il-6 reporter cells were infected with M. smegmatis at MOIs of 3:1, 10:1, and 30:1 (Fig. 4D). GFP was detected in the macrophages 24 h postinfection at all three MOIs, with the optimal signal being detected at an MOI of 30:1. Such experiments established the usefulness of this reporter cell line to monitor an innate inflammatory response upon exposure to mycobacteria.

Previous work has shown that pathogenic mycobacteria cause an attenuated inflammatory response compared to nonpathogenic strains (7). This is partly attributed to the release of ESAT-6 by strains such as M. tuberculosis (33). M. smegmatis clones that expressed high levels of ESAT-6 were tested for their ability to inhibit the IL-6 promoter-driven GFP expression in J774A.1 cell lines. Macrophages were infected at MOIs of 3:1, 10:1, and 30:1 with either the M. smegmatis parental cells or the clones expressing ESAT-6. The levels of GFP, as measured by flow cytometry, were indistinguishable at each MOI tested, regardless of whether the macrophages were infected with M. smegmatis clones lacking or containing the kan-MCS2-esat-6 expression vector (Fig. 5A). These experiments suggested one of two possibilities. First, ESAT-6 needed to be secreted to suppress innate inflammatory pathways. Second, ESAT-6 does not inhibit Il-6 promoter activity. To address these possibilities, pure ESAT-6 was added to the Il-6-gfp macrophage reporter cell line, either alone or with LPS or M. smegmatis (Fig. 5B). Unexpectedly, pure ESAT-6 directly activated the Il-6-gfp reporter cell line, as measured by the increased fluorescence intensity (Fig. 5B, upper). Moreover, the activation of the IL-6 reporter cell line was stronger in the LPS-stimulated cultures when ESAT-6 was supplemented. The macrophage cell line was clearly activated, as revealed by increased forward and side scatter (Fig. 5B, lower). In fact, the fluorescence levels were higher in the presence of pure ESAT-6 than with LPS (Fig. 5C). The ability of ESAT-6 to induce this strong inflammatory response was reduced when combined with M. smegmatis, matching the GFP expression when the macrophages were exposed to the mycobacteria. We speculate that this is due to ESAT-6 sequestration by the mycobacteria. To examine if the activation of the IL-6 pathway correlated with ESAT-6 levels, a dose-response assay was undertaken. Starting at 12 μg/ml of pure ESAT-6, a serial dilution indicated that levels as low as 50 ng/ml induced GFP expression by around 25-fold (Fig. 5D). These experiments indicate that ESAT-6 can stimulate certain components of the innate inflammatory response.

Fig 5
ESAT-6 activates innate inflammatory responses in macrophages through the IL-6-driven inflammatory pathway. (A) M. smegmatis clones expressing esat-6 were used to infect the IL-6 macrophage reporter cell line at MOIs of 3:1, 10:1, and 30:1. The mean fluorescence ...


The genomic sequence of M. tuberculosis was completed in 1998, revealing a complex genetic organization with approximately 4,000 open reading frames (12). However, relatively few gene products have been identified that directly attenuate innate immune cell functions (6, 19, 24, 27, 28, 33, 34, 39, 42, 45). Several distinct approaches have been used to identify putative virulence factors, including transposon mutagenesis of M. tuberculosis, isolation and characterization of regions of difference (RD), and studies of eukaryotic-like proteins encoded by the M. tuberculosis genome. This has led to the identification of a few virulence factors. However, none can fully explain the ability of M. tuberculosis to maintain a latent infection in infected humans without overtly activating the innate inflammatory response. For example, the suppressive effects of ESAT-6, a well-known virulence factor, require μg quantities in in vitro systems, and some studies have suggested that ESAT-6 can increase inflammatory responses (11, 30, 33). One limitation of these studies is a system amenable to high-level protein expression in infected cells. We re-engineered the mycobacterial shuttle vector pSUM36 to provide high-level protein expression in bacterial cultures and in infected macrophages. By using the kanamycin cassette already present in pSUM36 and incorporating a Shine-Dalgarno sequence downstream of this cassette, a 50-fold improvement in GFP expression was achieved compared to that obtained with the original lacZα promoter. The pSUM-kan vector was suitable for expressing GFP and ESAT-6 in E. coli and the mycobacterial strains M. smegmatis and M. avium. The levels of protein expression were consistently higher in the mycobacterial strains. This enabled us to readily detect the GFP-expressing mycobacteria within macrophages. An interesting difference in bacterial growth/morphology was uncovered with high-resolution confocal microscopy. The M. avium clones appeared as discrete foci within the macrophages. This was in marked contrast to the M. smegmatis clones, which exhibited a filamentous pattern that resembled spider webs within the cell. The basis for these differences is under investigation. The high levels of GFP expressed by the mycobacteria did not attenuate the growth or virulence of the bacteria. This is in contrast to a recent report that M. marinum virulence is reduced in a zebra fish infection model when GFP is expressed to high levels (31). The differences may relate to our use of macrophages in in vitro assays compared to the in vivo infections in the latter study.

A second useful tool developed in the current study was a macrophage reporter cell line responsive to bacterial lipopolysaccharide and M. smegmatis infections. This stable cell line provides a 40-fold range of GFP expression in every cell infected. Another report described the use of immunoreporter macrophage cell lines (6). In that case, the macrophages only provided transient reporter expression that had a wide fluorescence distribution. The current cell line provides sensitive, uniform GFP expression in all cells in response to mycobacterial infections. We tested the M. smegmatis clones overexpressing ESAT-6 to determine how ESAT-6 suppresses innate inflammatory pathways. Importantly, this intracellular form of ESAT-6 did not suppress an innate inflammatory response, as measured via the activation of the IL-6 promoter through either LPS or M. smegmatis. Since a previous report suggested that ESAT-6 must be released in a secreted form to attenuate Toll-like receptor signaling, we tested pure ESAT-6 in our IL-6 reporter assay (33). ESAT-6 was purified using established procedures that eliminate contaminating LPS. Surprisingly, ESAT-6 strongly activated the IL-6-dependent inflammatory pathway, as indicated by a 40-fold increase in GFP expression. Our data are consistent with another published report that ESAT-6 induces a strong IL-6 and tumor growth factor response (11). The mechanism by which ESAT-6 attenuates Toll-like receptor signaling, as reported elsewhere, requires further clarification but is not supported by our data. It is interesting that the combination of pure ESAT-6 with M. smegmatis caused GFP expression that was lower than that with just ESAT-6 alone. This could be due to ESAT-6 sequestration by M. smegmatis, although further experiments are required to address this possibility. In summary, our new protein expression vectors and reporter cell line provide additional tools for uncovering the pathogenic mechanisms of diverse mycobacterial strains. For example, this modified vector provides investigators with an excellent tool for testing different drugs for the eradication of M. avium and/or M. tuberculosis from infected cells (41). In addition, such a vector could enhance immune responses to specific mycobacterial proteins, which may further improve the efficacy of the recently developed M. smegmatis vaccines (43).

Supplementary Material

Supplemental material:


We thank Maria Labandeira-Rey and Dana Dodd for their helpful suggestions, critiques, and reagents. We also thank Angela Mobley for assistance with flow cytometry using mycobacterial species. We appreciate the help of Amie Torres, Sarah Gonzales, and Sara Ireland in testing the mycobacterial plasmids. The ESAT clones were obtained from Colorado State University and are currently provided by BEI Resources.

This work was supported in part by High Risk/High Impact grants from the University of Texas Southwestern Medical Center to N.S.C.V.O. and National Institutes of Health/National Institute of General Medical Sciences Director New Innovator Award 1 DP2 OD001886 to T.G.


Published ahead of print 20 July 2012

Supplemental material for this article may be found at


1. Aagaard C, et al. 2011. A multistage tuberculosis vaccine that confers efficient protection before and after exposure. Nat. Med. 17: 189–194 [PubMed]
2. Ainsa JA, Martin C, Cabeza M, De la Cruz F, Mendiola MV. 1996. Construction of a family of Mycobacterium/Escherichia coli shuttle vectors derived from pAL5000 and pACYC184: their use for cloning an antibiotic-resistance gene from Mycobacterium fortuitum. Gene 176: 23–26 [PubMed]
3. Andersen P, Doherty TM. 2005. The success and failure of BCG—implications for a novel tuberculosis vaccine. Nat. Rev. Microbiol. 3: 656–662 [PubMed]
4. Baccam M, Woo SY, Vinson C, Bishop GA. 2003. CD40-mediated transcriptional regulation of the IL-6 gene in B lymphocytes: involvement of NF-kappa B, AP-1, and C/EBP. J. Immunol. 170: 3099–3108 [PubMed]
5. Basaraba RJ, Izzo AA, Brandt L, Orme IM. 2006. Decreased survival of guinea pigs infected with Mycobacterium tuberculosis after multiple BCG vaccinations. Vaccine 24: 280–286 [PubMed]
6. Beaulieu AM, et al. 2010. Genome-wide screen for Mycobacterium tuberculosis genes that regulate host immunity. PLoS One 5: e15120 doi:10.1371/journal.pone.0015120 [PMC free article] [PubMed]
7. Beltan E, Horgen L, Rastogi N. 2000. Secretion of cytokines by human macrophages upon infection by pathogenic and non-pathogenic mycobacteria. Microb. Pathog. 28: 313–318 [PubMed]
8. Bertholet S, et al. 2010. A defined tuberculosis vaccine candidate boosts BCG and protects against multidrug-resistant Mycobacterium tuberculosis. Sci. Transl. Med. 2: 53ra74 doi:10.1126/scitranslmed.3001094 [PMC free article] [PubMed]
9. Brosch R, et al. 2002. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc. Natl. Acad. Sci. U. S. A. 99: 3684–3689 [PubMed]
10. Carlsson F, et al. 2010. Host-detrimental role of Esx-1-mediated inflammasome activation in mycobacterial infection. PLoS Pathog. 6: e1000895 doi:10.1371/journal.ppat.1000895 [PMC free article] [PubMed]
11. Chatterjee S, et al. 2011. Early secreted antigen ESAT-6 of Mycobacterium tuberculosis promotes protective T helper 17 cell responses in a toll-like receptor-2-dependent manner. PLoS Pathog. 7: e1002378 doi:10.1371/journal.ppat.1002378 [PMC free article] [PubMed]
12. Cole ST, et al. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393: 537–544 [PubMed]
13. Converse SE, Cox JS. 2005. A protein secretion pathway critical for Mycobacterium tuberculosis virulence is conserved and functional in Mycobacterium smegmatis. J. Bacteriol. 187: 1238–1245 [PMC free article] [PubMed]
14. Corbett EL, et al. 2003. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch. Intern. Med. 163: 1009–1021 [PubMed]
15. Cosma CL, Sherman DR, Ramakrishnan L. 2003. The secret lives of the pathogenic mycobacteria. Annu. Rev. Microbiol. 57: 641–676 [PubMed]
16. Davis JM, Ramakrishnan L. 2009. The role of the granuloma in expansion and dissemination of early tuberculous infection. Cell 136: 37–49 [PMC free article] [PubMed]
17. de Jonge MI, et al. 2007. ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity. J. Bacteriol. 189: 6028–6034 [PMC free article] [PubMed]
18. Dhandayuthapani S, et al. 1995. Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages. Mol. Microbiol. 17: 901–912 [PubMed]
19. Dussurget O, et al. 2001. Role of Mycobacterium tuberculosis copper-zinc superoxide dismutase. Infect. Immun. 69: 529–533 [PMC free article] [PubMed]
20. Fortune SM, et al. 2005. Mutually dependent secretion of proteins required for mycobacterial virulence. Proc. Natl. Acad. Sci. U. S. A. 102: 10676–10681 [PubMed]
21. Guinn KM, et al. 2004. Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol. Microbiol. 51: 359–370 [PMC free article] [PubMed]
22. Hsu T, et al. 2003. The primary mechanism of attenuation of bacillus Calmette Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc. Natl. Acad. Sci. USA 100: 12420–12425 [PubMed]
23. Kaufmann SH. 2010. Future vaccination strategies against tuberculosis: thinking outside the Box. Immunity 33: 567–577 [PubMed]
24. Koul A, et al. 2000. Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis. J. Bacteriol. 182: 5425–5432 [PMC free article] [PubMed]
25. Kremer L, Baulard A, Estaquier J, Poulain-Godefroy O, Locht C. 1995. Green fluorescent protein as a new expression marker in mycobacteria. Mol. Microbiol. 17: 913–922 [PubMed]
26. Lopez B, et al. 2003. A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes. Clin. Exp. Immunol. 133: 30–37 [PubMed]
27. Malik ZA, et al. 2003. Cutting edge: Mycobacterium tuberculosis blocks Ca2+ signaling and phagosome maturation in human macrophages via specific inhibition of sphingosine kinase. J. Immunol. 170: 2811–2815 [PubMed]
28. Manca C, Paul S, Barry CE, III, Freedman VH, Kaplan G. 1999. Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro. Infect. Immun. 67: 74–79 [PMC free article] [PubMed]
29. Miller WG, Lindow SE. 1997. An improved GFP cloning cassette designed for prokaryotic transcriptional fusions. Gene 191: 149–153 [PubMed]
30. Mishra BB, et al. 2010. Mycobacterium tuberculosis protein ESAT-6 is a potent activator of the NLRP3/ASC inflammasome. Cell. Microbiol. 12: 1046–1063 [PubMed]
31. Mutoji KN, Ennis DG. 2012. Expression of common fluorescent reporters may modulate virulence for Mycobacterium marinum: dramatic attenuation results from Gfp over-expression. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 155: 39–48 [PubMed]
32. Pasipanodya JG, et al. 2007. Pulmonary impairment after tuberculosis. Chest 131: 1817–1824 [PubMed]
33. Pathak SK, et al. 2007. Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. Nat. Immunol. 8: 610–618 [PubMed]
34. Raghavan S, Manzanillo P, Chan K, Dovey C, Cox JS. 2008. Secreted transcription factor controls Mycobacterium tuberculosis virulence. Nature 454: 717–721 [PMC free article] [PubMed]
35. Ramon-Garcia S, Otal I, Martin C, Gomez-Lus R, Ainsa JA. 2006. Novel streptomycin resistance gene from Mycobacterium fortuitum. Antimicrob. Agents Chemother. 50: 3920–3922 [PMC free article] [PubMed]
36. Renshaw PS, et al. 2002. Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1 complex and characterization of the structural properties of ESAT-6, CFP-10, and the ESAT-6-CFP-10 complex. Implications for pathogenesis and virulence. J. Biol. Chem. 277: 21598–21603 [PubMed]
37. Schagger H, von Jagow G. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166: 368–379 [PubMed]
38. Shaw JE, Pasipanodya JG, Gumbo T. 2010. Meningeal tuberculosis: high long-term mortality despite standard therapy. Medicine 89: 189–195 [PubMed]
39. Singh R, et al. 2003. Disruption of mptpB impairs the ability of Mycobacterium tuberculosis to survive in guinea pigs. Mol. Microbiol. 50: 751–762 [PubMed]
40. Smith J, et al. 2008. Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole. Infect. Immun. 76: 5478–5487 [PMC free article] [PubMed]
41. Srivastava S, et al. 2010. Efflux-pump-derived multiple drug resistance to ethambutol monotherapy in Mycobacterium tuberculosis and the pharmacokinetics and pharmacodynamics of ethambutol. J. Infect. Dis. 201: 1225–1231 [PMC free article] [PubMed]
42. Stanley SA, Raghavan S, Hwang WW, Cox JS. 2003. Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc. Natl. Acad. Sci. U. S. A. 100: 13001–13006 [PubMed]
43. Sweeney KA, et al. 2011. A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis. Nat. Med. 17: 1261–1268 [PMC free article] [PubMed]
44. Volkman HE, et al. 2010. Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium. Science 327: 466–469 [PMC free article] [PubMed]
45. Walburger A, et al. 2004. Protein kinase G from pathogenic mycobacteria promotes survival within macrophages. Science 304: 1800–1804 [PubMed]

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