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Mycobacterium tuberculosis ESX-1 system-secreted protein ESAT-6 but not CFP10 inhibits human T-cell immune responses
Tuberculosis 89 (2009) S1, S74–S76
Contents lists available at ScienceDirect
Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube
Mycobacterium tuberculosis ESX-1 system-secreted protein ESAT-6 but not CFP10 inhibits human T-cell immune responses Buka Samtena,b, *, Xisheng Wanga,b , Peter F. Barnesa,b,c a Center
for Pulmonary and Infectious Disease Control, b Departments of Microbiology and Immunology, and c Medicine, University of Texas Health Science Center, Tyler, TX 75708, USA
article info
summary
Keywords: Human Cytokine Transcription factor ESAT-6 Tuberculosis
The secreted proteins of M. tuberculosis, early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP10), have been identified as antigenic proteins with potent T-cell stimulatory effects, and therefore have been the focus of tuberculosis vaccine studies. However, recent work showed that secretion of these proteins by the specialized ESAT-6 secretion system (ESX)-1 of M. tuberculosis is associated with virulence and pathogenesis. The studies demonstrated that ESAT-6 inhibits antigen-presenting cell function by reducing IL-12 production by macrophages through interrupting TLR2 signaling pathways and inducing macrophage apoptosis. However, the effect of ESAT-6 on T cells remains unexplored. To address this question, we studied the effect of recombinant ESAT-6 and CFP10 on human primary T-cell IFN-g secretion and proliferation. ESAT-6, but not CFP10, inhibited IFN-g production by T cells stimulated with M. tuberculosis or with antiCD3 plus anti-CD28, in a dose-dependent manner. ESAT-6 also inhibited T-cell production of IL-17 and TNF-a, but not IL-2. Presence of CFP10 as part of the ESAT-6/CFP10 heterodimer did not affect ESAT-6 inhibition of T-cell IFN-g production. ESAT-6 inhibited the proliferation of CD3+ cells in response to TCR stimulation. ESAT-6 decreased T-cell IFN-g secretion by mechanisms independent of cytotoxicity or apoptosis. ESAT-6 reduced IFN-g mRNA levels by inhibiting the expression of the transcription factors, ATF-2, c-Jun and CREB, which upregulate IFN-g gene expression in T cells through binding to the IFN-g proximal promoter. ESAT-6, but not CFP10, bound to T cells and inhibited expression of early activation markers without reducing phosphorylation of ZAP70, a proximal TCR signaling molecule. We conclude that ESAT-6 directly inhibits human T-cell responses by affecting TCR signaling pathways downstream of ZAP70. © 2009 Elsevier Ltd. All rights reserved.
1. ESAT-6 as a vaccine antigen Early secreted antigenic target of 6 kD (ESAT-6) was originally purified from the short-term culture filtrate proteins of M. tuberculosis H37Rv, and induced strong T-cell recall responses in M. tuberculosis-infected mice.1,2 Studies in different species of mycobacteria revealed that esat-6 and its product ESAT-6 were absent in all the attenuated strains of M. bovis BCG but exist in virulent M. bovis and all M. tuberculosis strains.3 To understand the gene regulation of esat-6 (Rv3875) in M. tuberculosis, researchers identified a gene(Rv3874), located immediate upstream of esat-6, that was cotranscribed with esat-6, and encoded a 10 kD protein, designated CFP10, that also exists in the short-term culture filtrate proteins of M. tuberculosis.4 Due to their strong immunogenicity and relatively restricted distribution in pathogenic mycobacteria, ESAT-6 and CFP10 have been considered putative vaccine proteins.5 In addition, two new tuberculosis diagnostic tests, QuantiFERON-TB * Corresponding author. Buka Samten, CPIDC, UT Health Science Center, 11937 US Hwy 271, Tyler, TX, 75708-3154, USA. Tel.: +1 (903) 877 7665; fax: +1 (903) 877 7989. E-mail address: [email protected] (B. Samten). 1472-9792 /$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
Gold (QTF-G) and T-SPOT.TB, have been developed to measure the immune response to these proteins for the diagnosis of latent and active tuberculosis infection.6 2. ESAT-6 as a virulence factor Although ESAT-6 and CFP10 have been recognized as antigenic proteins with vaccine potential, recent studies have clearly demonstrated that secretion of these two proteins is associated with virulence and pathogenicity of tuberculosis, based on studies in macrophages and in vivo in M. tuberculosis-infected animals. Genomic analysis identified three major regions of difference (RD1 to RD3) which were deleted in avirulent M. bovis BCG strains, compared to the virulent M. bovis and M. tuberculosis strains.7 Further studies found that the genes encoding ESAT-6 and CFP10 are located within RD1.8 Reintroduction of RD1 restored virulence to M. bovis BCG, suggesting that RD1-encoded genes cause virulence and pathogenicity of M. tuberculosis. The recent discovery of the specialized ESAT-6 secretion system (ESX)-1, which is responsible for secretion of ESAT-6 and CFP10 as a heterodimer,9 further strengthened their association with virulence by demonstrating
B. Samten et al. / Tuberculosis 89 (2009) S74–S76
that active secretion of ESAT-6 and CFP10 by ESX-1 is required for development of tuberculosis infection in animals.10–12 Inhibition of IL-12 secretion by M. tuberculosis-infected macrophages is associated with active secretion of ESAT-6,12,13 providing the first evidence that ESX-1-mediated secretion of ESAT-6 and CFP10 may subvert the host immune responses to M. tuberculosis infection. Studies have demonstrated that recombinant ESAT-6 inhibits monocyte IL-12 secretion by interrupting TLR2 signaling pathways,14 lending further support for this association. However, the direct effects of ESAT-6 and CFP10 on T cells remain unclear. 3. ESAT-6 inhibits IFN-g production by T cells We recently made the surprising finding that ESAT-6 can directly inhibit IFN-g production by T cells.15 We produced recombinant ESAT-6 and CFP10, using Escherichia coli that had been transformed with plasmids containing inserts encoding ESAT-6 and CFP10. The level of lipopolysaccharide (LPS) in these protein preparations was very low. Peripheral blood mononuclear cells (PBMC) from QTF-G-positive healthy subjects were incubated with 0.8–3.3 mM ESAT-6 or CFP10 for 48 hours, or with 2 mg/ml whole washed M. tuberculosis Erdman (M. tb) as a positive control. M. tb, all concentrations of CFP10 and 0.8 mM of ESAT-6 induced comparable levels of IFN-g production, as measured by ELISA. However, 1.6 and 3.3 mM of ESAT-6 failed to induce IFN-g production, suggesting that its immunogenicity was reduced at higher concentrations. This was not due to increased IL-10 production, as assessed by ELISA. Next, we treated PBMC from QFT-G-positive healthy donors with different concentrations of recombinant ESAT-6 or CFP10 for one hour prior to stimulation with M. tb. PBMC treated with 1.6 mM of ESAT-6 had decreased M. tb-induced IFN-g secretion, and 3.3 mM of ESAT-6 inhibited IFN-g levels by more than 95%. These ESAT-6 concentrations also inhibited IFN-g production by purified CD3+ T cells that were stimulated by anti-CD3 and anti-CD28, indicating that this effect was independent of monocytes. Treatment of cells with ESAT-6/CFP10 heterodimers did not affect ESAT-6 inhibition of IFN-g. ESAT-6 (1.6 and 3.3 mM) also strongly inhibited IFN-g mRNA expression by PBMC stimulated with M. tb and T cells stimulated with anti-CD3 and anti-CD28. Depletion of ESAT-6 from the recombinant preparations with a nickel column abrogated inhibition of IFN-g production by CD3+ T cells, demonstrating that this was not due to contaminants in the ESAT-6 preparation. 4. ESAT-6 inhibits other T-cell functions but does not cause cell death or apoptosis We found that ESAT-6 (1.6 and 3.3 mM), but not CFP10, strongly inhibited proliferation of T cells stimulated with anti-CD3 and antiCD28, as measured by CFSE dilution.15 ESAT-6 reduced expression of the early T-cell activation markers, CD25 and CD69. In addition, ESAT-6 inhibited production of TNF-a and IL-17 by T cells stimulated with anti-CD3 and anti-CD28. However, it did not inhibit IL-2 production, indicating that ESAT-6 was not a cellular toxin and that its effects were specific for certain cytokines. ESAT-6 did not affect T-cell viability, as assessed by Trypan blue staining and the MTT assay, and had no effect on apoptosis, measured by annexin V staining and flow cytometry. 5. ESAT-6 binds to T cells and inhibits transcription factors that bind to the IFN-g promoter Using biotinylated ESAT-6 and CFP10 and flow cytometry, we recently found that ESAT-6 binds to CD4+ and CD8+ T cells, as well as CD14+ monocytes and CD19+ B cells, whereas CFP10 binds only to CD14+ and CD19+ cells in PBMC. This indicates that ESAT-6 directly binds to human primary T cells.15 We also found that treatment of
S75
M. tb-stimulated PBMC with 3.3 mM of ESAT-6 reduced expression of the transcription factors, cyclic AMP response element binding protein (CREB), activating transcription factor (ATF)-2 and c-Jun activating protein (AP)-1, which are known to upregulate IFN-g gene transcription through binding to the proximal promoter of IFN-g.16,17 As an initial step to understand ESAT-6 inhibition of T-cell activation, we studied the effect of ESAT-6 on phosphorylation of the proximal T-cell activation signaling molecule, ZAP70,18 in response to crosslinking of TCR with anti-CD3 mAb and goat anti-mouse IgG. The results from this study demonstrated that ESAT-6 did not affect phosphorylation of ZAP-70, suggesting that ESAT-6 does not affect the T-cell immune synapse or the proximal molecules associated with TCR signaling. We believe that ESAT-6 affects the molecules downstream of ZAP-70, and work is underway to dissect these mechanisms more fully. 6. Conclusion Our recent findings described here demonstrate that the ESX-1 system-secreted ESAT-6 protein of M. tuberculosis directly binds to human primary T cells and inhibits IFN-g production in response to TCR stimulation through inhibiting expression of transcription factors that regulate IFN-g gene transcription. The mechanisms are not due to cell toxicity, apoptosis or inhibition of proximal TCR signaling molecules, and remain to be addressed in the future. We speculate that ESAT-6-induced inhibition of IFN-g production may play a role in subversion of host immune responses by M. tuberculosis. Acknowledgements: These studies were supported by Grants from the National Institutes of Health [A1063514], the James Byers Cain Research Endowment, and the Center for Pulmonary and Infectious Disease Control. This work was presented in part at the Texas Tuberculosis Research Symposium (TTRS) 2009, Houston, TX, cosponsored by the University of Texas Health Sciences CenterHouston and the Methodist Hospital Research Institute. Competing interests: The authors have no financial conflict of interest. References 1. Andersen P, Andersen AB, Sorensen AL, Nagai S. Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J Immunol 1995;154:3359–72. 2. Sorensen AL, Nagai S, Houen G, Andersen P, Andersen AB. Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infect Immun 1995;63:1710–7. 3. Harboe M, Oettinger T, Wiker HG, Rosenkrands I, Andersen P. Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG. Infect Immun 1996;64:16–22. 4. Berthet FX, Rasmussen PB, Rosenkrands I, Andersen P, Gicquel B. A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10). Microbiology (Reading) 1998;144(Pt 11):3195– 203. 5. Brodin P, Rosenkrands I, Andersen P, Cole ST, Brosch R. ESAT-6 proteins: protective antigens and virulence factors? Trends Microbiol 2004;12:500–8. 6. Pai M, Zwerling A, Menzies D. Systematic review: T-cell-based assays for the diagnosis of latent tuberculosis infection: an update. Ann Intern Med 2008;149: 177–84. 7. Mahairas GG, Sabo PJ, Hickey MJ, Singh DC, Stover CK. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 1996;178:1274–82. 8. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998;393:537–44. 9. Renshaw PS, Panagiotidou P, Whelan A, Gordon SV, Hewinson RG, Williamson RA, et al. 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 2002;277:21598–603.
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10. Guinn KM, Hickey MJ, Mathur SK, Zakel KL, Grotzke JE, Lewinsohn DM, et al. Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol Microbiol 2004;51:359–70. 11. Hsu T, Hingley-Wilson SM, Chen B, Chen M, Dai AZ, Morin PM, et al. 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 2003;100:12420–5. 12. Stanley SA, Raghavan S, Hwang WW, Cox JS. Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc Natl Acad Sci USA 2003;100:13001–6. 13. Stanley SA, Johndrow JE, Manzanillo P, Cox JS. The Type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. J Immunol 2007;178:3143–52. 14. Pathak SK, Basu S, Basu KK, Banerjee A, Pathak S, Bhattacharyya A, et al.
15.
16.
17.
18.
Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. Nat Immunol 2007;8:610–8. Wang X, Barnes PF, Dobos-Elder KM, Townsend JC, Chung Y-t, Shams H, et al. ESAT-6 inhibits production of IFN-g by Mycobacterium tuberculosis-responsive human T cells. J Immunol 2009;182:3668–77. Samten B, Howard ST, Weis SE, Wu S, Shams H, Townsend JC, et al. Cyclic AMP response element-binding protein positively regulates production of IFN-gamma by T cells in response to a microbial pathogen. J Immunol 2005;174:6357–63. Samten B, Townsend JC, Weis SE, Bhoumik A, Klucar P, Shams H, et al. CREB, ATF, and AP-1 transcription factors regulate IFN-gamma secretion by human T cells in response to mycobacterial antigen. J Immunol 2008;181:2056–64. Wange RL, Samelson LE. Complex complexes: signaling at the TCR. Immunity 1996;5:197–205.
Contents lists available at ScienceDirect
Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube
Mycobacterium tuberculosis ESX-1 system-secreted protein ESAT-6 but not CFP10 inhibits human T-cell immune responses Buka Samtena,b, *, Xisheng Wanga,b , Peter F. Barnesa,b,c a Center
for Pulmonary and Infectious Disease Control, b Departments of Microbiology and Immunology, and c Medicine, University of Texas Health Science Center, Tyler, TX 75708, USA
article info
summary
Keywords: Human Cytokine Transcription factor ESAT-6 Tuberculosis
The secreted proteins of M. tuberculosis, early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP10), have been identified as antigenic proteins with potent T-cell stimulatory effects, and therefore have been the focus of tuberculosis vaccine studies. However, recent work showed that secretion of these proteins by the specialized ESAT-6 secretion system (ESX)-1 of M. tuberculosis is associated with virulence and pathogenesis. The studies demonstrated that ESAT-6 inhibits antigen-presenting cell function by reducing IL-12 production by macrophages through interrupting TLR2 signaling pathways and inducing macrophage apoptosis. However, the effect of ESAT-6 on T cells remains unexplored. To address this question, we studied the effect of recombinant ESAT-6 and CFP10 on human primary T-cell IFN-g secretion and proliferation. ESAT-6, but not CFP10, inhibited IFN-g production by T cells stimulated with M. tuberculosis or with antiCD3 plus anti-CD28, in a dose-dependent manner. ESAT-6 also inhibited T-cell production of IL-17 and TNF-a, but not IL-2. Presence of CFP10 as part of the ESAT-6/CFP10 heterodimer did not affect ESAT-6 inhibition of T-cell IFN-g production. ESAT-6 inhibited the proliferation of CD3+ cells in response to TCR stimulation. ESAT-6 decreased T-cell IFN-g secretion by mechanisms independent of cytotoxicity or apoptosis. ESAT-6 reduced IFN-g mRNA levels by inhibiting the expression of the transcription factors, ATF-2, c-Jun and CREB, which upregulate IFN-g gene expression in T cells through binding to the IFN-g proximal promoter. ESAT-6, but not CFP10, bound to T cells and inhibited expression of early activation markers without reducing phosphorylation of ZAP70, a proximal TCR signaling molecule. We conclude that ESAT-6 directly inhibits human T-cell responses by affecting TCR signaling pathways downstream of ZAP70. © 2009 Elsevier Ltd. All rights reserved.
1. ESAT-6 as a vaccine antigen Early secreted antigenic target of 6 kD (ESAT-6) was originally purified from the short-term culture filtrate proteins of M. tuberculosis H37Rv, and induced strong T-cell recall responses in M. tuberculosis-infected mice.1,2 Studies in different species of mycobacteria revealed that esat-6 and its product ESAT-6 were absent in all the attenuated strains of M. bovis BCG but exist in virulent M. bovis and all M. tuberculosis strains.3 To understand the gene regulation of esat-6 (Rv3875) in M. tuberculosis, researchers identified a gene(Rv3874), located immediate upstream of esat-6, that was cotranscribed with esat-6, and encoded a 10 kD protein, designated CFP10, that also exists in the short-term culture filtrate proteins of M. tuberculosis.4 Due to their strong immunogenicity and relatively restricted distribution in pathogenic mycobacteria, ESAT-6 and CFP10 have been considered putative vaccine proteins.5 In addition, two new tuberculosis diagnostic tests, QuantiFERON-TB * Corresponding author. Buka Samten, CPIDC, UT Health Science Center, 11937 US Hwy 271, Tyler, TX, 75708-3154, USA. Tel.: +1 (903) 877 7665; fax: +1 (903) 877 7989. E-mail address: [email protected] (B. Samten). 1472-9792 /$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
Gold (QTF-G) and T-SPOT.TB, have been developed to measure the immune response to these proteins for the diagnosis of latent and active tuberculosis infection.6 2. ESAT-6 as a virulence factor Although ESAT-6 and CFP10 have been recognized as antigenic proteins with vaccine potential, recent studies have clearly demonstrated that secretion of these two proteins is associated with virulence and pathogenicity of tuberculosis, based on studies in macrophages and in vivo in M. tuberculosis-infected animals. Genomic analysis identified three major regions of difference (RD1 to RD3) which were deleted in avirulent M. bovis BCG strains, compared to the virulent M. bovis and M. tuberculosis strains.7 Further studies found that the genes encoding ESAT-6 and CFP10 are located within RD1.8 Reintroduction of RD1 restored virulence to M. bovis BCG, suggesting that RD1-encoded genes cause virulence and pathogenicity of M. tuberculosis. The recent discovery of the specialized ESAT-6 secretion system (ESX)-1, which is responsible for secretion of ESAT-6 and CFP10 as a heterodimer,9 further strengthened their association with virulence by demonstrating
B. Samten et al. / Tuberculosis 89 (2009) S74–S76
that active secretion of ESAT-6 and CFP10 by ESX-1 is required for development of tuberculosis infection in animals.10–12 Inhibition of IL-12 secretion by M. tuberculosis-infected macrophages is associated with active secretion of ESAT-6,12,13 providing the first evidence that ESX-1-mediated secretion of ESAT-6 and CFP10 may subvert the host immune responses to M. tuberculosis infection. Studies have demonstrated that recombinant ESAT-6 inhibits monocyte IL-12 secretion by interrupting TLR2 signaling pathways,14 lending further support for this association. However, the direct effects of ESAT-6 and CFP10 on T cells remain unclear. 3. ESAT-6 inhibits IFN-g production by T cells We recently made the surprising finding that ESAT-6 can directly inhibit IFN-g production by T cells.15 We produced recombinant ESAT-6 and CFP10, using Escherichia coli that had been transformed with plasmids containing inserts encoding ESAT-6 and CFP10. The level of lipopolysaccharide (LPS) in these protein preparations was very low. Peripheral blood mononuclear cells (PBMC) from QTF-G-positive healthy subjects were incubated with 0.8–3.3 mM ESAT-6 or CFP10 for 48 hours, or with 2 mg/ml whole washed M. tuberculosis Erdman (M. tb) as a positive control. M. tb, all concentrations of CFP10 and 0.8 mM of ESAT-6 induced comparable levels of IFN-g production, as measured by ELISA. However, 1.6 and 3.3 mM of ESAT-6 failed to induce IFN-g production, suggesting that its immunogenicity was reduced at higher concentrations. This was not due to increased IL-10 production, as assessed by ELISA. Next, we treated PBMC from QFT-G-positive healthy donors with different concentrations of recombinant ESAT-6 or CFP10 for one hour prior to stimulation with M. tb. PBMC treated with 1.6 mM of ESAT-6 had decreased M. tb-induced IFN-g secretion, and 3.3 mM of ESAT-6 inhibited IFN-g levels by more than 95%. These ESAT-6 concentrations also inhibited IFN-g production by purified CD3+ T cells that were stimulated by anti-CD3 and anti-CD28, indicating that this effect was independent of monocytes. Treatment of cells with ESAT-6/CFP10 heterodimers did not affect ESAT-6 inhibition of IFN-g. ESAT-6 (1.6 and 3.3 mM) also strongly inhibited IFN-g mRNA expression by PBMC stimulated with M. tb and T cells stimulated with anti-CD3 and anti-CD28. Depletion of ESAT-6 from the recombinant preparations with a nickel column abrogated inhibition of IFN-g production by CD3+ T cells, demonstrating that this was not due to contaminants in the ESAT-6 preparation. 4. ESAT-6 inhibits other T-cell functions but does not cause cell death or apoptosis We found that ESAT-6 (1.6 and 3.3 mM), but not CFP10, strongly inhibited proliferation of T cells stimulated with anti-CD3 and antiCD28, as measured by CFSE dilution.15 ESAT-6 reduced expression of the early T-cell activation markers, CD25 and CD69. In addition, ESAT-6 inhibited production of TNF-a and IL-17 by T cells stimulated with anti-CD3 and anti-CD28. However, it did not inhibit IL-2 production, indicating that ESAT-6 was not a cellular toxin and that its effects were specific for certain cytokines. ESAT-6 did not affect T-cell viability, as assessed by Trypan blue staining and the MTT assay, and had no effect on apoptosis, measured by annexin V staining and flow cytometry. 5. ESAT-6 binds to T cells and inhibits transcription factors that bind to the IFN-g promoter Using biotinylated ESAT-6 and CFP10 and flow cytometry, we recently found that ESAT-6 binds to CD4+ and CD8+ T cells, as well as CD14+ monocytes and CD19+ B cells, whereas CFP10 binds only to CD14+ and CD19+ cells in PBMC. This indicates that ESAT-6 directly binds to human primary T cells.15 We also found that treatment of
S75
M. tb-stimulated PBMC with 3.3 mM of ESAT-6 reduced expression of the transcription factors, cyclic AMP response element binding protein (CREB), activating transcription factor (ATF)-2 and c-Jun activating protein (AP)-1, which are known to upregulate IFN-g gene transcription through binding to the proximal promoter of IFN-g.16,17 As an initial step to understand ESAT-6 inhibition of T-cell activation, we studied the effect of ESAT-6 on phosphorylation of the proximal T-cell activation signaling molecule, ZAP70,18 in response to crosslinking of TCR with anti-CD3 mAb and goat anti-mouse IgG. The results from this study demonstrated that ESAT-6 did not affect phosphorylation of ZAP-70, suggesting that ESAT-6 does not affect the T-cell immune synapse or the proximal molecules associated with TCR signaling. We believe that ESAT-6 affects the molecules downstream of ZAP-70, and work is underway to dissect these mechanisms more fully. 6. Conclusion Our recent findings described here demonstrate that the ESX-1 system-secreted ESAT-6 protein of M. tuberculosis directly binds to human primary T cells and inhibits IFN-g production in response to TCR stimulation through inhibiting expression of transcription factors that regulate IFN-g gene transcription. The mechanisms are not due to cell toxicity, apoptosis or inhibition of proximal TCR signaling molecules, and remain to be addressed in the future. We speculate that ESAT-6-induced inhibition of IFN-g production may play a role in subversion of host immune responses by M. tuberculosis. Acknowledgements: These studies were supported by Grants from the National Institutes of Health [A1063514], the James Byers Cain Research Endowment, and the Center for Pulmonary and Infectious Disease Control. This work was presented in part at the Texas Tuberculosis Research Symposium (TTRS) 2009, Houston, TX, cosponsored by the University of Texas Health Sciences CenterHouston and the Methodist Hospital Research Institute. Competing interests: The authors have no financial conflict of interest. References 1. Andersen P, Andersen AB, Sorensen AL, Nagai S. Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J Immunol 1995;154:3359–72. 2. Sorensen AL, Nagai S, Houen G, Andersen P, Andersen AB. Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infect Immun 1995;63:1710–7. 3. Harboe M, Oettinger T, Wiker HG, Rosenkrands I, Andersen P. Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG. Infect Immun 1996;64:16–22. 4. Berthet FX, Rasmussen PB, Rosenkrands I, Andersen P, Gicquel B. A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10). Microbiology (Reading) 1998;144(Pt 11):3195– 203. 5. Brodin P, Rosenkrands I, Andersen P, Cole ST, Brosch R. ESAT-6 proteins: protective antigens and virulence factors? Trends Microbiol 2004;12:500–8. 6. Pai M, Zwerling A, Menzies D. Systematic review: T-cell-based assays for the diagnosis of latent tuberculosis infection: an update. Ann Intern Med 2008;149: 177–84. 7. Mahairas GG, Sabo PJ, Hickey MJ, Singh DC, Stover CK. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 1996;178:1274–82. 8. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998;393:537–44. 9. Renshaw PS, Panagiotidou P, Whelan A, Gordon SV, Hewinson RG, Williamson RA, et al. 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 2002;277:21598–603.
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10. Guinn KM, Hickey MJ, Mathur SK, Zakel KL, Grotzke JE, Lewinsohn DM, et al. Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol Microbiol 2004;51:359–70. 11. Hsu T, Hingley-Wilson SM, Chen B, Chen M, Dai AZ, Morin PM, et al. 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 2003;100:12420–5. 12. Stanley SA, Raghavan S, Hwang WW, Cox JS. Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc Natl Acad Sci USA 2003;100:13001–6. 13. Stanley SA, Johndrow JE, Manzanillo P, Cox JS. The Type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. J Immunol 2007;178:3143–52. 14. Pathak SK, Basu S, Basu KK, Banerjee A, Pathak S, Bhattacharyya A, et al.
15.
16.
17.
18.
Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. Nat Immunol 2007;8:610–8. Wang X, Barnes PF, Dobos-Elder KM, Townsend JC, Chung Y-t, Shams H, et al. ESAT-6 inhibits production of IFN-g by Mycobacterium tuberculosis-responsive human T cells. J Immunol 2009;182:3668–77. Samten B, Howard ST, Weis SE, Wu S, Shams H, Townsend JC, et al. Cyclic AMP response element-binding protein positively regulates production of IFN-gamma by T cells in response to a microbial pathogen. J Immunol 2005;174:6357–63. Samten B, Townsend JC, Weis SE, Bhoumik A, Klucar P, Shams H, et al. CREB, ATF, and AP-1 transcription factors regulate IFN-gamma secretion by human T cells in response to mycobacterial antigen. J Immunol 2008;181:2056–64. Wange RL, Samelson LE. Complex complexes: signaling at the TCR. Immunity 1996;5:197–205.