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Mycobacterium tuberculosis (Mtb) causes death of 2–3 million people annually and is ... 98–106; doi:10.1038/sj.icb.7100117; published online 2 October 2007.
Immunology and Cell Biology (2008) 86, 98–106 & 2008 Australasian Society for Immunology Inc. All rights reserved 0818-9641/08 $30.00 www.nature.com/icb

ORIGINAL ARTICLE

Mycobacterium tuberculosis secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex inhibit lipopolysaccharide-induced NF-jB transactivation by downregulation of reactive oxidative species (ROS) production Niladri Ganguly1, Pham H Giang1, Chitra Gupta2, Sandip K Basu1, Imran Siddiqui1, Dinakar M Salunke2 and Pawan Sharma1 Mycobacterium tuberculosis (Mtb) causes death of 2–3 million people annually and is considered one of the most successful intracellular pathogens to persist inside the host macrophage. Recent studies have implicated the role of RD-1 region of Mtb genome in the mycobacterial pathogenesis. The role of RD-1-encoded secretory proteins of Mtb in modulation of macrophage function has not been investigated in detail. Here we show that RD-1 encoded two major secretory proteins, namely, culture filtrate protein-10 kDa (CFP-10) and early secreted antigenic target-6 kDa (ESAT-6), and their 1:1 CFP-10:ESAT6 complex inhibit production of reactive oxidative species (ROS) in RAW264.7 cells. These proteins also downregulated the bacterial lipopolysaccharide (LPS)-induced ROS production, which, in turn, downregulated LPS-induced nuclear factor-jB (NF-jB) p65 DNA-binding activity, as well as inhibited the NF-jB-dependent reporter gene (chloramphenicol acetyl transferase) expression in the treated macrophages. Moreover, addition of N-acetyl cysteine, which is a scavenger of ROS, also inhibited LPS-induced reporter gene expression by scavenging the ROS, thereby preventing NF-jB transactivation. These studies indicate that the secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex of Mtb can inhibit LPS-induced NF-jB-dependent gene expression via downregulation of ROS production. Immunology and Cell Biology (2008) 86, 98–106; doi:10.1038/sj.icb.7100117; published online 2 October 2007 Keywords: CFP-10; ESAT-6; lipopolysaccharide; NF-kB; ROS

Tuberculosis is a global health problem caused by Mycobacterium tuberculosis (Mtb) with 2–3 million deaths per year.1 The transmission of the infection occurs primarily through respiratory tract by inhalation of fine droplets containing the bacilli. The naive or resting macrophages are extremely prone to invasion by Mtb bacilli and are unable to mount any antimycobacterial response associated with activated macrophages.2–6 Thus, the resting macrophage seems to provide an ideal niche where intracellular tubercle bacilli may reside, replicate and persist.7 The proteins secreted by Mtb during its sojourn through macrophage have gained attention in recent years both as vaccine candidates as well as virulence factors.8–17 Some of them like culture filtrate protein-10 kDa (CFP-10) and early secreted antigenic target-6 kDa (ESAT-6) are encoded by the RD-1 region of Mtb genome and are absent in all the bacillus Calmette-Guerin (BCG) strains.18–21

Generation of reactive oxidative species (ROS) by macrophages constitute an important arm of antibacterial response.22 ROS are a group of reactive oxygen species that include oxygen anions and radicals (O2 and OH) or the milder oxidants such as hydrogen peroxide (H2O2). H2O2 oxidizes the sulfhydryl (–SH) group of cysteine to sulfenic acid, which is readily reduced to cysteine by various cellular reducing agents, including glutathione and thioredoxin. The fact that only certain proteins inside the cell carry an oxidizable cysteine at a critical position is the reason why such a small molecule like H2O2 can act as a specific second messenger. Our studies show that RD-1 encoded antigens CFP-10 and ESAT-6 as well as the CFP10:ESAT6 complex23–26 downregulated ROS production in RAW264.7 macrophages. Lipopolysaccharide (LPS) is known to induce ROS production in macrophages.27–29 ROS activates IkB

1Immunology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India and 2Structural Biology Laboratory, National Institute of Immunology, New Delhi, India Correspondence: Dr Pawan Sharma, Immunology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, PO Box-10504, New Delhi 110067, India. E-mail: [email protected] Received 1 June 2007; revised 21 July 2007; accepted 16 August 2007; published online 2 October 2007

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kinase leading to the removal of inhibitory IkB subunit from the nuclear factor-kB (NF-kB); the latter then translocates to the nucleus and binds to its cognate DNA-binding site and induces transcription of genes.30–34 We rationalized that the effect of CFP-10, ESAT-6 and CFP10:ESAT6 on ROS production would modulate NF-kB p65 subunit activation, DNA-binding and transactivation property. It could be a mechanism where the secretory proteins of Mtb can modulate the macrophage signaling pathways. RESULTS The authenticity of recombinant proteins was ascertained by nucleotide sequencing of the recombinant clones, by the western blot analysis of the expressed and purified recombinant proteins, their amino-acid analysis and finally by N-terminal amino-acid sequencing of the purified recombinant ESAT-6 and CFP-10 preparations. The western blots of CFP-10 and ESAT-6 are given as the Supplementary Figures S1, S2, which clearly show that the recombinant proteins being expressed were of expected size. The N-terminal sequence of the purified recombinant proteins was determined for their first 30 residues by Edman degradation method using Applied Biosystem’s Procise sequencer (Applied Biosystems, Foster City, CA, USA). The N-terminal protein sequence for recombinant ESAT-6 was found to be MTEQQWNFAGIEAAASAIQGNVTSIHSLLD, and for CFP-10 it was MRGSHHHHHHTDPMAEMKTDAATLAQEAGN. It may be pertinent to point out here that while our recombinant CFP-10 was heat stable as reported earlier,35 we found that recombinant ESAT-6 was quite heat labile and tended to undergo autoproteolysis if not stored properly as noted by Renshaw and colleagues.23 The structural states of CFP-10, ESAT-6 and CFP10:ESAT6 complex were confirmed under the experimental conditions by far-UV circular dichroism (CD) spectroscopy. The far-UV CD spectra shown in Figure 1 are representative of those obtained for ESAT-6, CFP-10 and CFP10:ESAT6 complex, respectively. The spectra for ESAT-6 and the CFP10:ESAT6 complex are similar to proteins that have significant amount of helical content, whereas a strikingly different spectrum

Figure 1 The circular dichroism spectra of CFP-10 (A), ESAT-6 (B) and the 1:1 CFP10:ESAT6 complex (C) measured in a Jasco J 500 polarimeter. The CD spectra were collected from protein samples dissolved in buffer 25 mM NaH2PO4, 100 mM NaCl, pH 6.5. Spectra were recorded from 190 to 250 nm at a scan speed of 50 nm min 1, each spectrum representing an average of 10 accumulations. During acquisition of spectra, the samples were maintained at 18 1C in a 0.1 mm path length cell.

observed for CFP-10 indicates unstructured, random coil polypeptide. These findings are similar to those reported earlier by Renshaw and colleagues.23 The complex formation was also indicated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and nativePAGE analysis of the three entities (Supplementary Figures S3, S4). The SDS-PAGE showed that in the complex both CFP-10 and ESAT-6 were present in equal amounts. In the native-PAGE the complex came up at higher position compared to ESAT-6 and showed a single band, which indicated that the two proteins were in a complexed form. The higher band of CFP-10 in the native-PAGE gel was likely due to the formation of oligomers under native conditions.23 Inhibition of ROS production by CFP-10, ESAT-6 and CFP10:ESAT6 complex Measurement of ROS production in RAW264.7 cells showed that all the three components CFP-10, ESAT-6 and CFP10:ESAT6 complex downregulated ROS over 5 min of treatment. The downregulation observed with CFP10:ESAT6 complex was more than that observed with CFP-10 or ESAT-6 alone. The complex at 5 mg ml 1 decreased ROS by about 60% vis-a`-vis 40% reduction seen with ESAT-6, and 10% reduction observed with CFP-10 compared to that observed in the unstimulated cells (Figure 2). To check for the specificity of this effect, we used two non-ESAT-6 family mycobacterial-secreted proteins, namely, CFP-21 and Antigen 85B, at a similar dose, for stimulating the RAW264.7 cells and measured ROS production (Figure 3); these proteins had no significant effect on the ROS levels as compared to those observed in the unstimulated cells. In order to determine whether this effect was restricted to RAW264.7 cells only, we checked the levels of ROS induced by CFP-10, ESAT-6 and the complex in the mouse peritoneal macrophages (Figure 4) as well as in the J774A.1 cells (see Supplementary Figure S5), and we found that in these latter cell populations as well, all the three constructs reduced ROS production as compared to that seen in the unstimulated cells. Next, we wanted to see the effect of these proteins on the LPSinduced ROS production. CFP-10 inhibited the LPS-induced ROS production in RAW cells by 60% (Figure 5), ESAT-6 by 50%, while the CFP10:ESAT6 complex decreased the LPS-induced ROS production by 70%. The CFP-21 on the other hand did not have any appreciable effect on the LPS-induced ROS production. The effect of LPS on ROS production (Figure 5) appears to be rather weak (B30% increase in

Figure 2 Effect of CFP-10, ESAT-6 and CFP10:ESAT6 complex on reactive oxidative species (ROS) generation. ROS production as measured by 10 mM H2DCFDA oxidation are shown. Briefly, H2DCFDA-labeled RAW264.7 were plated at 3105 per well of a 96-well plate, and the fluorescence signal obtained was recorded for 5 min at 37 1C. The production of ROS by stimulation of RAW264.7 cells with 5, 10 and 20 mg ml 1 of CFP-10, ESAT-6 and the CFP10:ESAT6 complex. Data represented as mean±s.d. of three independent experiments. Immunology and Cell Biology

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Figure 3 The inhibition of reactive oxidative species (ROS) production is specific for ESAT-6 family proteins. RAW264.7 macrophages were treated with different doses of two non-ESAT-6 family proteins, CFP-21 and Antigen 85B. The graph shows the generation of ROS as measured by H2DCFDA oxidation upon stimulation with 5, 10 and 20 mg ml 1 of CFP-21 and Antigen-85B for 5 min at 37 1C. The data are represented as mean±s.d. of three independent experiments.

Figure 4 The effect of CFP-10, ESAT-6 and CFP10:ESAT6 complex on reactive oxidative species (ROS) production is observed with primary macrophages also and is not limited to RAW264.7 cells. Mouse peritoneal macrophages were loaded with H2DCFDA as before and fluorescence signals recorded for 5 min at 37 1C. The graph shows the change in the production of ROS in mouse peritoneal macrophages obtained from C57/BL6 mice upon stimulation with 5, 10 and 20 mg ml 1 of CFP-10, ESAT-6 and the CFP10:ESAT6 complex. The data are representatives of two independent experiments (** indicates significant difference (Po0.01) over unstimulated cells).

ROS from the baseline). However, it is pertinent to point out that this data was obtained very early after stimulation with LPS, that is, 5 min after addition of LPS. Others have also shown almost similar level of effect of LPS in macrophages at comparable time point and dose.36–38 Inhibition of LPS-induced NF-jB p65 DNA-binding activity by CFP-10, ESAT-6 and the CFP10:ESAT6 complex LPS is a known inducer of ROS production and the latter activates transcription factor NF-kB leading to gene expression.30–34 Since CFP10, ESAT-6 and CFP10:ESAT6 complex antagonized the ROS production in macrophages, we determined the effect of inhibition of ROS production on the NF-kB p65 DNA-binding activity in the nucleus as a measure of its activation. For this RAW264.7 cells were treated with LPS (0.1 mg ml 1) and/or CFP-10 (5 mg ml 1), ESAT-6 (5 mg ml 1), the complex (5 mg ml 1) and N-acetyl cysteine (10 mM) for 30 min. A control was set up where 100-fold excess of nonradioactive (cold) probe was added along with labeled probe. In another set, anti-p65 Immunology and Cell Biology

Figure 5 CFP-10, ESAT-6 and CFP10:ESAT6 complex antagonized lipopolysaccharide (LPS)-induced reactive oxidative species (ROS) production in RAW264.7 cells. For this cells were stimulated with LPSand/or CFP-10, ESAT-6 and the complex for 5 min at 37 1C and ROS was measured as before using 10 mM H2DCFDA. The graph shows the change in ROS production upon different stimulations. Lane 1, unstimulated cells; lane 2, cells+LPS (0.1 mg ml 1); lane 3, cells+CFP-10 (5 mg ml 1); lane 4, cells+LPS (0.1 mg ml 1)+CFP-10 (5 mg ml 1); lane 5, cCells+ESAT-6 (5 mg ml 1); lane 6, cells+LPS (0.1 mg ml 1)+ESAT-6 (5 mg ml 1); lane 7, cells+CFP10:ESAT6 complex (5 mg ml 1); lane 8, cells+LPS (0.1 mg ml 1)+CFP10:ESAT6 complex (5 mg ml 1); lane 9, cells+CFP-21 (5 mg ml 1); lane 10, cells+LPS (0.1 mg ml 1)+CFP-21 (5 mg ml 1). The data is represented as mean±s.d. of three independent experiments (*indicates significant (Po0.05) difference over LPS-stimulated cells).

antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was added in the binding reaction. The control where 100-fold excess of unlabeled probe was added showed no binding of p65 (Figure 6a). The control where anti-p65 antibody was added showed inhibition of binding of p65 to DNA; it has been reported that inhibition of DNA– protein complexation by antibody also indicates specificity.39 We found that LPS induced a twofold increase in the p65 DNA-binding activity over the basal level, while CFP-10 and ESAT-6 antagonized this effect back to the basal level. Interestingly, the CFP10:ESAT6 complex further modulated the LPS effect, causing decrease in p65 DNAbinding activity by 40% compared to the basal levels observed in the unstimulated cells (Figure 6b). As expected, the addition of N-acetyl cysteine (10 mM), a known antioxidant, also inhibited LPS induction of p65 DNA-binding activity by 60% compared to that in the unstimulated cells. These observations corroborated the role of ROS in the activation of NF-kB p65 and its subsequent DNA-binding activity; since the addition of N-acetyl cysteine removes the ROS species, it counters the positive effect of LPS on p65 and hence the binding of p65 subunit falls below the basal level.40,41 Inhibition of LPS-induced NF-jB-dependent reporter gene expression by CFP-10, ESAT-6 and CFP10:ESAT6 complex Since LPS is known to induce ROS production and the latter activates transcription factor NF-kB leading to gene expression, RAW264.7 cells were transfected with a reporter plasmid containing chloramphenicol acetyl transferase (cat) gene under interleukin-2 (IL-2) promoter (which contains binding sites for NF-kB p65 subunit), and cells were stimulated with different stimulating agents for 2 h. The autoradiogram obtained after doing the chloramphenicol acetyltransferase (CAT) assay is shown in Figure 7a. LPS treatment upregulated cat expression by 80% over the basal level as shown in Figure 7b. The LPSinduced increase was downregulated by CFP-10, ESAT-6 and CFP10:ESAT6 complex by 100, 70 and 90%, respectively (Figure 7b)

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Figure 6 The downregulation of lipopolysaccharide (LPS)-induced ROS production by CFP-10, ESAT-6 and CFP10:ESAT6 complex inhibit LPS-induced nuclear factor-kB (NF-kB) p65 DNA-binding activity. RAW264.7 cells were stimulated with LPS and/or CFP-10, ESAT-6, CFP10:ESAT6 complex and N-acetyl cysteine (NAC, 10 mM) for 30 min, nuclear extracts obtained was probed with a labeled NF-kB consensus binding sequence and run on a polyacrylamide gel and exposed to X-ray film. (a) Autoradiogram of the gel after doing electrophoretic mobility shift assay. Lane 1, unstimulated cells; lane 2, LPS (0.1 mg ml 1); lane 3, LPS+CFP10 (5 mg ml 1); lane 4, LPS+ESAT6 (5 mg ml 1); lane 5, LPS+CFP10:ESAT6 complex (5 mg ml 1); lane 6, CFP-10 (5 mg ml 1); lane 7, ESAT-6 (5 mg ml 1); lane 8, CFP10:ESAT6 complex (5 mg ml 1); lane 9, LPS+N-acetyl cysteine (10 mM); lane 10, 100-fold excess (5 pmol) of unlabeled probe compared to labeled probe (50 fmol); lane 11, antibody control having 1 mg of anti-p65 antibody. The autoradiogram is a representative of two independent experiments. (b) The graph shows the plot of densitometric values obtained from the densitometric studies of the autoradiogram. Lanes 1–9 as mentioned above (**indicates significant difference (Po0.01) over LPS-stimulated cells).

compared to LPS stimulation alone. Stimulation with LPS along with N-acetyl cysteine (10 mM) also led to a significant reduction (120%) in cat expression as expected. These results indicated that inhibition of LPS-induced ROS production by these proteins adversely affected the NF-kB transcriptional activity. DISCUSSION The data presented here show that the mycobacterial secretory proteins CFP-10, ESAT-6 and their complex interfered with the LPS-induced production of ROS by the macrophages, leading to an inhibition of the NF-kB transactivation property. Others have earlier shown that ESAT-6 could indeed block innate immune responses; thus, Mtb mutants deficient in ESAT-6 secretion have been shown to enhance production of tumor necrosis factor-a (TNF-a) and IL-12,42 while engagement of TLR2 by ESAT-6 was found to dampen the MyD88-dependent arm of Toll-like receptor (TLR) signaling, leading

to inhibition of NF-kB activation.43 The present study indicates yet another possible mechanism by which ESAT-6 could exert its inhibitory effect on NF-kB activation, viz., via modulation of ROS response. Indeed, the generation of ROS has been associated with the stress response, apoptosis, ageing and cell death44,45 However, in intramacrophage infections, ROS seems to play a significant role in the host defense mechanism against bacteria by oxidizing the –SH group of cysteine in the proteins. Previous studies showed that ROS production in J774A.1 macrophages was markedly inhibited by addition of a nicotinamide adenine dinucleotide phosphate-oxidase inhibitor, diphenylene iodonium and the antioxidant N-acetyl cysteine (NAC).46 These observation suggest a mechanism by which binding of the protein to the cell surface might accelerate the process of breakdown of H2O2 into water and oxygen thereby reducing the level of available H2O2 in the cell. M. tuberculosis unlike other intracellular bacteria like Escherichia coli and Salmonella typhimurium are not Immunology and Cell Biology

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Figure 7 The inhibition of lipopolysaccharide (LPS)-induced NF-kB p65 DNA-binding activity by CFP-10, ESAT-6 and CFP10:ESAT6 complex downregulated NF-kB-dependent reporter gene, chloramphenicol acetyl transferase (cat) expression. RAW264.7 cells were stimulated with LPS and/or CFP-10, ESAT-6, complex and N-acetyl cysteine (NAC, 10 mM) for 120 min; cell extracts were prepared and the CAT assay done using 14C-chloramphenicol. The products of the CAT assay were run on a thin-layer chromatography plate using methanol:chloroform (95:5) as a solvent and the plate was exposed to X-ray film. (a) The autoradiogram shows di-acetylated chloramphenicol on the upper row, the monoacetylated chloramphenicol in the middle row and unacetylated chloramphenicol in the lower row. Lane 1, unstimulated cells; lane 2, LPS (0.1 mg ml 1); lane 3, LPS+ESAT-6 (5 mg ml 1); lane 4, LPS+CFP-10 (5 mg ml 1); lane 5, LPS+CFP10:ESAT6 complex (5 mg ml 1); lane 6, ESAT-6 (5 mg ml 1); lane 7, CFP-10 (5 mg ml 1); lane 8, CFP10:ESAT6 (5 mg ml 1); lane 9, LPS+Nacetyl cysteine (10 mM). The autoradiogram is a representative of three independent experiments. (b) The graph shows the change in densitometric values obtained after densitometric studies of the upper row (di-acetylated chloramphenicol) of the autoradiogram. Lanes 1–9 as mentioned above.

equipped to produce Oxy R, a critical component of oxidative stress response. Mtb does possess a host of components to potentially deal with ROS or oxidative stress, such as catalase peroxidase, KatG47–49 and two superoxide dismutase proteins, SodA and SodC.50 ROS is known to activate the transcription factor NF-kB that leads to transcription of genes.30–34 In the present study, LPS was found to induce the expression of NF-kB-dependent reporter gene cat, which was downregulated by all the three moieties. Addition of NAC, which is an ROS scavenger,40,41 along with LPS led to a significant reduction in the reporter gene expression confirming the role of ROS in the NFkB-dependent transcription. The transcription factor NF-kB plays a role in containment of infection by inducing the expression of several proinflammatory cytokines like TNF-a, IL-12, interferon-g (IFN-g) and nitric-oxide synthase 2,51–53 therefore it is critically targeted by pathogens. It has been reported that secretion of ESAT-6/CFP-10 requires proteins encoded by Snm 1, -2 and -4 of the RD-1 region, since mutations in these genes lead to a defect in the bacterial growth during acute phase of mouse infection. The Snm mutants fail to Immunology and Cell Biology

replicate in the in vitro macrophage culture as well as fail to inhibit macrophage inflammatory response.21,54–57 In a recent study, ESAT-6 has been shown to bind to the TLR-2 on the surface of macrophages and cause inhibition of activation of transcription factors NF-kB and interferon regulatory factors through the Akt kinase pathway.43 In another recent study in the dendritic cells, the phagolysosomes containing M. tuberculosis or M. leprae were found to be progressively translocated into the cytosol,58 and this translocation was dependent on the secretion of CFP-10 and ESAT-6. No such phenomenon was observed with M. bovis BCG or heat-killed mycobacteria. Furthermore, ESAT-6 has also been implicated in apoptosis of macrophages through a caspase-dependent pathway,59 as well as in lysis of the infected cells.60,61 However, in our study, we did not observe any such effect of ESAT-6 on the cell viability during the experimental time period; we routinely monitored cell viability in such experiments in terms of mitochondrial respiration, with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, as mentioned in one of our earlier papers.35 Also, we did not observe

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any apoptosis of macrophages as measured by annexinV-fluorescein isothyocyanate and propidium iodide staining (data not presented). The interplay between mycobacteria and host immunity can be decisive in determining the outcome of infection. Previous studies from our group have focused on the effect of CFP-10 and ESAT-6 on macrophage and dendritic cell functions.62–64 Others have shown that CFP-10 and ESAT-6 can activate mast cells as well.65 We have previously shown that CFP-10 induces TNF-a production,35 and our unpublished data show that ESAT-6 also induces release of TNF-a by the macrophages. Although TNF-a is known to be involved in granuloma formation which helps in containment of the Mtb bacilli, however, early TNF-a secretion could also promote replication of bacilli inside the macrophages.66,67 Moreover, secretory proteins may also participate in sustained TNF-a secretion accompanying persistence of mycobacteria inside the macrophages, leading to tissue destruction by necrosis resulting in dissemination of the bacilli. It was reported that administration of mycobacterial antigens to mice with prior infection by Mtb resulted in exacerbation of lung pathology through TNF-a-induced inflammation.68 Mtb-specific secretory proteins, therefore, might play a dual role in the immunity against tuberculosis—although apparently indispensable for the induction of the protective response as others have shown,9–12 they could also contribute to its subversion in predisposed individuals, which could be partly mediated through interference with macrophage and dendritic cell function as results presented here as well as those in our previous work have demonstrated. Our studies show that the CFP-10, ESAT-6 and the 1:1 CFP10:ESAT6 complex downregulated the NF-kBdependent reporter gene expression through inhibition of ROS production in RAW264.7 cells. This perhaps represents a mechanism by which the secretory proteins of Mtb might help in creating a favorable niche inside the macrophages and thereby contribute to the mycobacterial pathogenesis. Further work with mutant Mtb deficient in ESAT-6 and/orCFP-10 might yield a more detailed insight into the mechanisms by which this modulation of macrophage function is achieved. METHODS Reagents and antibodies Bacterial LPS and NAC and other fine chemicals were obtained from Sigma (St Louis, MO, USA). Tissue culture medium Dulbecco’s modified Eagle’s medium (DMEM) and the antibiotics penicillin and streptomycin and fetal bovine serum (FBS) were obtained from Life Technologies (Carlsbad, CA, USA).

Polyacrylamide gel electrophoresis of proteins The SDS-PAGE of proteins was performed in the presence of 0.1% SDS in the gels. Protein samples were prepared by mixing with equal volume of 2 SDSPAGE sample buffer and boiling in a sand bath for 10 min. Gel was run at a constant voltage of 200 V. The native-PAGE gels for the proteins were run with tris-borate-ethylenediaminetetraacetic acid as running buffer in the absence of SDS. Protein samples were prepared by mixing 2 PAGE loading dye (without SDS and b-mercaptoethanol) and kept on ice. The gel was run at 200 V at 4 1C.

Western blotting For western blotting of proteins mini Trans-blot Electrophoretic Cell (Bio-Rad, Hercules, CA, USA) was used to transfer the proteins from gel onto nitrocellulose membrane. The apparatus for electroblotting was assembled according to the manufacturer’s instructions. Electroblotting was performed at a constant current of 150 mA for 1 h. Following which the membrane was incubated in blocking buffer for 2 h with gentle shaking at room temperature. After that, the blocking solution was discarded and after washing twice with wash buffer the blot was incubated with an appropriate dilution of primary anti-polyhistidine

antibody (1:2000) for another 2 h with gentle shaking. Thereafter, the blot was washed thrice with wash buffer for 5 min each. After washing, the blot was incubated with horseradish peroxidase-conjugated secondary antibody (1:2000) solution for 2 h. The blot was washed as described above and processed using diamino-benzidine and 30% H2O2. Blocking buffer—5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) (0.1% Tween-20) Wash buffer—PBS (0.1% Tween-20)

Silver staining of polyacrylamide gels After the gel was run, the proteins were fixed by incubating the gel in fixing solution (methanol:acetic acid:water, 50:5:45) for 30 min. The gel was rinsed with water (two changes, 2 min per change) and then left in water for 1 h on a shaking platform. Extended washing was done to eliminate yellowish background usually observed after overdeveloping of the gel. The gel was sensitized with sensitizing solution (0.02% sodium thiosulfate) for 2 min and the solution was discarded with a quick rinse of the gel with two changes of water (10 s each). Chilled silver-nitrate solution (0.1% AgNO3) was added and the gel was kept for 30 min at 4 1C in dark. Silver-nitrate solution was discarded and the gel was quickly rinsed with two changes of water (30 s per each change). The gel was developed with developing solution (0.04% formaldehyde in 2% sodium carbonate) and as soon as it turned yellow, it was replaced with fresh solution. When sufficient degree of staining was obtained, the developing solution was discarded and replaced by stop solution (1% acetic acid) to quench the staining. The gel was then washed several times with water and dried.

Circular dichroism spectroscopy The far-UV CD spectra was used to determine the secondary structure of ESAT6, CFP-10 and ESAT6:CFP10 complex in a Jasco J 500 polarimeter. The CD spectra were collected from protein samples dissolved in buffer 25 mM NaH2PO4, 100 mM NaCl, pH 6.5. Spectra were recorded from 190 to 250 nm at a scan speed of 50 nm min 1, each spectrum representing an average of 10 accumulations. During acquisition of spectra, the samples were maintained at 18 1C in a 0.1 mm path length cell.

Expression of ESAT-6 protein E. coli BL21(DE3)pLysS transformed with pET23b(+) (Novagen, Madison, WI, USA) vector were grown in Luria Bertani (LB) medium containing 100 mg ml 1 of ampicillin and 34 mg ml 1 of chloramphenicol (Amersham Biosciences, Piscataway, NJ, USA). The expression of ESAT-6 was induced at mid-log phase (corresponding to an absorbance of 0.6 at 600 nm) by the addition of isopropyl-1-thio-b-D-galactopyranoside to 0.45 mM for 4 h at 37 1C. After this cells were harvested by centrifugation at 5000 r.p.m. for 15 min at 4 1C. The cell pellets were lysed in 10 mM NaH2PO4 and 0.3 M NaCl, pH 8.0 containing 1 mg ml 1 each of aprotinin, leupeptin, pepstatin A and 1 mg ml 1 of lysozyme. The cells were sonicated four times by giving a pulse of 40 s each. The suspension was then centrifuged at 13 000 r.p.m. for 20 min at 4 1C and the inclusion bodies obtained as pellets were dissolved in 8 M Urea pH 8.0 at room temperature. Then the suspension was centrifuged at 13 000 r.p.m. for 30 min at room temperature. The lysate was then allowed to bind with Ni2-NTA resin (Qiagen, Valencia, CA, USA) and then the flow through collected. The pure fractions of ESAT-6 were pooled and dialyzed initially against 10 mM Na2HPO4, pH 8.0 to remove urea and then dialyzed against several changes of refolding buffer 25 mM NaH2PO4 and 100 mM NaCl, pH 6.5. The endotoxin level in the protein was determined using E-Toxate kit (Sigma) and did not exceed 0.03 endotoxin units in any batch of the purified recombinant ESAT-6 protein. The N-terminal sequencing of the expressed protein was done by Edman degradation method using Applied Biosystem’s Procise sequencer. The N-terminal sequencing of purified recombinant ESAT-6 yielded amino-acid sequence for 30 residues as MTEQQWNFAGIEAAASAIQGNVTSIHSLLD, which matches completely with the published sequence of ESAT-6.

Expression of CFP-10 protein E. coli cells M15 transformed with pQE31(Novagen) vector was allowed to grow in LB medium containing 100 mg ml 1 of ampicillin and 25 mg ml 1 kanamycin. The expression of CFP-10 was induced in mid-log phase Immunology and Cell Biology

Secretory proteins on ROS production N Ganguly et al 104 (corresponding to an absorbance of 0.6 at 600 nm). After 4 h, cells were harvested by centrifugation at 5000 r.p.m. for 15 min at 4 1C. The pellet was then lysed in a buffer containing 10 mM NaH2PO4 and 0.3 M NaCl, pH 8.0 containing 1 mg ml 1 of aprotinin, leupeptin, pepstatin A and 1 mg ml 1 of lysozyme. Then the cells were sonicated by giving four pulses of 40 s each. Then the suspension was centrifuged at 13 000 r.p.m. for 20 min at 4 1C. The lysate was allowed to bind with Ni2+-NTA resin and then the flow through collected. The resin was then washed with phosphate buffer (10 mM NaH2PO4 and 0.3 M NaCl, pH 8.0) containing 20 and 250 mM imidazole, respectively. The pure fractions of protein were dialyzed against refolding buffer; 25 mM NaH2PO4 and 100 mM NaCl, pH 6.5. The endotoxin levels did not exceed 0.03 endotoxin units. The N-terminal sequence of our expressed CFP-10 protein was MRGSHHHHHHTDPMAEMKTDAATLAQEAGN.

Preparation of CFP-10:ESAT-6 complex For preparation of complex the protein content of the purified CFP-10 and ESAT-6 samples were measured using bicinchonic acid reagent with BSA as a standard. The 1:1 complex of CFP-10 and ESAT-6 was made by mixing equimolar amounts of CFP-10 and ESAT-6 in refolding buffer; 25 mM NaH2PO4 and 100 mM NaCl, pH 6.5 for 30 min at room temperature.

Isolation of peritoneal macrophages Mouse peritoneal macrophages were obtained from resident peritoneal cells of C57BL/6 mice. The animals were anesthetized and the resident peritoneal cells were harvested, pooled and counted with a hemocytometer. This cell suspension was adjusted to 1106 viable cells ml 1 in DMEM medium and incubated for 2 h at 37 1C. The adherent cells were collected and used for further experiments.

Maintenance of cell lines Murine macrophage cell lines RAW264.7 and J774A.1, originally obtained from ATCC (Manassas, VA, USA), were routinely maintained in DMEM containing 2 mM glutamine, 100 mg ml 1 of penicillin and streptomycin and 10% FBS at 5% CO2 in a humidified atmosphere at 37 1C.

Measurement of intracellular ROS For measurement of ROS production intracellularly, 3106 cells were loaded with 10 mM dichloro-dihydroxy fluorescein diacetate (DCFDA), for 10 min at 37 1C in serum-free DMEM. Then cells were washed twice with DMEM and then resuspended in 1 ml of DMEM, 100 ml of cell suspension was added per well of 96-well plate and ROS production was monitored in a spectrofluorimeter, FluoSTAR OPTIMA (BMG Technologies, Mount Eliza, VIC, Australia).

Electrophoretic mobility shift assay Electrophoretic mobility Shift Assay (EMSA) was done for NF-kB p65 subunit. After stimulation with respective stimuli, cells were harvested and lysed in 300 ml of lysis buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM phenylmethylsulphonylfluoride, 1 mM sodium orthovanadate (Na3VO4), 1 mM sodium fluoride, 1 mg ml 1 each of leupeptin, pepstatin A and aprotinin and 1% NP-40) for 20 min on ice. The cell lysates so obtained were cleared by centrifugation at 13 000 r.p.m., the supernatant represented the cytoplasmic extract; the nuclear pellet was washed and resuspended in the nuclear extraction buffer (20 mM HEPES, pH 7.9, 400 mM KCl, 10 mM EDTA, 10 mM EGTA) and kept on ice for 40 min with intermittent vortexing. Finally, the suspension was centrifuged at 13 000 r.p.m. at 4 1C, the supernatant was the nuclear extract. The protein content of nuclear extract was estimated by Bradford Reagent (Bio-Rad, USA) and used for binding reaction. A 32P-labeled NF-kB consensus binding sequence (underlined) was used as a probe for the EMSA reaction. NF-kB wt (sense): 5¢-AGTTGAGGGGACTTTCCCAGGC-3¢ NF-kB wt (anti): 3¢-GCCTGGGAAAGTCCCCTCAACT-5¢ Immunology and Cell Biology

Labeling of the DNA probe The labeling reaction was done at 37 1C for 20 min using 5 pmol of the probe. The volume of the reaction was made up to 200 ml with distilled water. The free nucleotides was removed from the 32P-labeled probe by Nucleotide Removal Kit (Qiagen). The labeled probe was eluted from the column by 125 ml of distilled water. For the EMSA binding reaction 10 mg of the total nuclear extract was used for binding to the labeled probe. The binding reaction was carried out on ice for 1 h. The reaction was stopped by using 4 ml of 6 DNA loading dye. The samples were then loaded on to a native-PAGE gel and run at 200 V for 1 h 30 min. The gel was then dried and exposed to the X-ray film for 12 h to get the autoradiogram. Composition The composition of 10 EMSA binding buffer—100 mM HEPES (pH 7.9), 5 mM EDTA (pH 8.0), 5 mM EGTA (pH 8.0), 5 mM dithiothreitol, 10 mM MgCl2, 40% glycerol and 1% NP-40.

Transfection and reporter gene assay For reporter assay, RAW264.7 cells were transfected with 5 mg of the reporter plasmid containing IL-2 promoter (which contain NF-kB p65 binding sites) downstream of which is the cat gene. Transfection was done in serum-free and antibiotic-free DMEM for 6 h using lipofectamine (Invitrogen Inc., Carlsbad, CA, USA) in 60 mm Petri dish (Nunc Inc., Roskilde, Denmark). After 6 h the cells were washed with PBS and complete DMEM was added, the cells were then stimulated for 2 h with appropriate stimuli as described in the ‘Results’ section. After 2 h the medium was aspirated and fresh complete medium was added and the cells were kept for 48 h. After 48 h cells were harvested, washed, lysed by repeated freeze thawing and the cell extract was obtained by centrifuging the suspension at 13 000 r.p.m. for 5 min at 4 1C. The cell extract was heat inactivated by heating at 65 1C for 10 min. Then assay was done using 14Cchloramphenicol and 20 ml of acetyl-CoASH for 3 h at 37 1C. Then the reaction product was extracted from the reaction mixture by adding 1 ml of ethyl acetate and dried and the product resuspended in 25 ml of ethyl acetate and then the samples were spotted and run on thin-layer chromatography plate using 5% methanol in chloroform as solvent. After this the plate was dried and exposed to X-ray film.

Statistical analysis Statistical analysis was performed using analysis of variance. Wherever F-value is significant (Po0.05, Po0.01), comparison has been done between means of samples and control. The * indicates significant differences (Po0.05) and ** indicates significant differences (Po0.01) over the control. Where F-value is insignificant it is not mentioned.

ACKNOWLEDGEMENTS NG and SKB are recipients of fellowships from CSIR and PHG is recipient of an international pre-doctoral fellowship from ICGEB. IS is supported through a DBT research grant to PS. The plasmid IL-2–cat was kindly provided by Dr Vijay Kumar, ICGEB, New Delhi.

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