Mycobacterium tuberculosis has evolved many strategies to evade elimination by the host immune system, including the selective repression of macrophage IL-12p40 production. To identify the M. tuberculosis genes responsible for this aspect of immune evasion, we used a macrophage cell line expressing a reporter for IL-12p40 transcription to screen a transposon library of M. tuberculosis for mutants that lacked this function. This approach led to the identification of the mmaA4 gene, which encodes a methyl transferase required for introducing the distal oxygen-containing modifications of mycolic acids, as a key locus involved in the repression of IL-12p40. Mutants in which mmaA4 (hma) was inactivated stimulated macrophages to produce significantly more IL-12p40 and TNF-α than wild-type M. tuberculosis and were attenuated for virulence. This attenuation was not seen in IL-12p40-deficient mice, consistent with a direct linkage between enhanced stimulation of IL-12p40 by the mutant and its reduced virulence. Treatment of macrophages with trehalose dimycolate (TDM) purified from the ΔmmaA4 mutant stimulated increased IL-12p40, similar to the increase observed from ΔmmaA4 mutant-infected macrophages. In contrast, purified TDM isolated from wild-type M. tuberculosis inhibited production of IL-12p40 by macrophages. These findings strongly suggest that M. tuberculosis has evolved mmaA4-derived mycolic acids, including those incorporated into TDM to manipulate IL-12-mediated immunity and virulence.
Flynn JL, Chan J (2003) Immune evasion by Mycobacterium tuberculosis: living with the enemy. Curr Opin Immunol 15: 450–455.
[4]
Tufariello JM, Chan J, Flynn JL (2003) Latent tuberculosis: mechanisms of host and bacillus that contribute to persistent infection. Lancet Infect Dis 3: 578–590.
[5]
Stead WW, Dutt AK (1991) Tuberculosis in elderly persons. Annu Rev Med 42: 267–276.
[6]
Ito K, Fujimori M, Shingu K, Hama Y, Kanai T, et al. (2005) Pulmonary tuberculosis in a patient receiving intensive chemotherapy for metastatic breast cancer. Breast J 11: 87–88.
Behr MA, Small PM (1997) Has BCG attenuated to impotence? Nature 389: 133–134.
[9]
Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O'Garra A, et al. (1993) Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260: 547–549.
[10]
Trinchieri G (2003) Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 3: 133–146.
[11]
Cooper AM, Magram J, Ferrante J, Orme IM (1997) Interleukin 12 (IL-12) is crucial to the development of protective immunity in mice intravenously infected with mycobacterium tuberculosis. J Exp Med 186: 39–45.
[12]
Holscher C, Atkinson RA, Arendse B, Brown N, Myburgh E, et al. (2001) A protective and agonistic function of IL-12p40 in mycobacterial infection. J Immunol 167: 6957–6966.
[13]
Khader SA, Pearl JE, Sakamoto K, Gilmartin L, Bell GK, et al. (2005) IL-23 compensates for the absence of IL-12p70 and is essential for the IL-17 response during tuberculosis but is dispensable for protection and antigen-specific IFN-gamma responses if IL-12p70 is available. J Immunol 175: 788–795.
[14]
Beadling C, Slifka MK (2006) Regulation of innate and adaptive immune responses by the related cytokines IL-12, IL-23, and IL-27. Arch Immunol Ther Exp (Warsz) 54: 15–24.
[15]
Khader SA, Partida-Sanchez S, Bell G, Jelley-Gibbs DM, Swain S, et al. (2006) Interleukin 12p40 is required for dendritic cell migration and T cell priming after Mycobacterium tuberculosis infection. J Exp Med 203: 1805–1815.
[16]
Feng CG, Jankovic D, Kullberg M, Cheever A, Scanga CA, et al. (2005) Maintenance of pulmonary Th1 effector function in chronic tuberculosis requires persistent IL-12 production. J Immunol 174: 4185–4192.
[17]
Altare F, Ensser A, Breiman A, Reichenbach J, Baghdadi JE, et al. (2001) Interleukin-12 receptor beta1 deficiency in a patient with abdominal tuberculosis. J Infect Dis 184: 231–236.
[18]
Jouanguy E, Doffinger R, Dupuis S, Pallier A, Altare F, et al. (1999) IL-12 and IFN-gamma in host defense against mycobacteria and salmonella in mice and men. Curr Opin Immunol 11: 346–351.
[19]
Hickman SP, Chan J, Salgame P (2002) Mycobacterium tuberculosis induces differential cytokine production from dendritic cells and macrophages with divergent effects on naive T cell polarization. J Immunol 168: 4636–4642.
[20]
Nau GJ, Richmond JF, Schlesinger A, Jennings EG, Lander ES, et al. (2002) Human macrophage activation programs induced by bacterial pathogens. Proc Natl Acad Sci U S A 99: 1503–1508.
[21]
Scott P, Hunter CA (2002) Dendritic cells and immunity to leishmaniasis and toxoplasmosis. Curr Opin Immunol 14: 466–470.
[22]
Carrera L, Gazzinelli RT, Badolato R, Hieny S, Muller W, et al. (1996) Leishmania promastigotes selectively inhibit interleukin 12 induction in bone marrow-derived macrophages from susceptible and resistant mice. J Exp Med 183: 515–526.
[23]
Greinert U, Ernst M, Schlaak M, Entzian P (2001) Interleukin-12 as successful adjuvant in tuberculosis treatment. Eur Respir J 17: 1049–1051.
[24]
Flynn JL, Goldstein MM, Triebold KJ, Sypek J, Wolf S, et al. (1995) IL-12 increases resistance of BALB/c mice to Mycobacterium tuberculosis infection. J Immunol 155: 2515–2524.
[25]
Dao DN, Kremer L, Guerardel Y, Molano A, Jacobs WR Jr, et al. (2004) Mycobacterium tuberculosis lipomannan induces apoptosis and interleukin-12 production in macrophages. Infect Immun 72: 2067–2074.
[26]
Manca C, Tsenova L, Barry CE 3rd, Bergtold A, Freeman S, et al. (1999) Mycobacterium tuberculosis CDC1551 induces a more vigorous host response in vivo and in vitro, but is not more virulent than other clinical isolates. J Immunol 162: 6740–6746.
[27]
Chacon-Salinas R, Serafin-Lopez J, Ramos-Payan R, Mendez-Aragon P, Hernandez-Pando R, et al. (2005) Differential pattern of cytokine expression by macrophages infected in vitro with different Mycobacterium tuberculosis genotypes. Clin Exp Immunol 140: 443–449.
[28]
Rubin EJ, Akerley BJ, Novik VN, Lampe DJ, Husson RN, et al. (1999) In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria. Proc Natl Acad Sci U S A 96: 1645–1650.
[29]
Bardarov S, Bardarov S Jr, Jr, Pavelka MS Jr, Jr, Sambandamurthy V, Larsen M, et al. (2002) Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 148: 3007–3017.
[30]
Pompei L, Jang S, Zamlynny B, Ravikumar S, McBride A, et al. (2007) Disparity in IL-12 release in dendritic cells and macrophages in response to Mycobacterium tuberculosis is due to use of distinct TLRs. J Immunol 178: 5192–5199.
[31]
Boissier F, Bardou F, Guillet V, Uttenweiler-Joseph S, Daffe M, et al. (2006) Further insight into S-adenosylmethionine-dependent methyltransferases: structural characterization of Hma, an enzyme essential for the biosynthesis of oxygenated mycolic acids in Mycobacterium tuberculosis. J Biol Chem 281: 4434–4445.
[32]
Dinadayala P, Laval F, Raynaud C, Lemassu A, Laneelle MA, et al. (2003) Tracking the putative biosynthetic precursors of oxygenated mycolates of Mycobacterium tuberculosis. Structural analysis of fatty acids of a mutant strain deviod of methoxy- and ketomycolates. J Biol Chem 278: 7310–7319.
[33]
Van Boxtel RM, Lambrecht RS, Collins MT (1990) Effects of colonial morphology and tween 80 on antimicrobial susceptibility of Mycobacterium paratuberculosis. Antimicrob Agents Chemother 34: 2300–2303.
[34]
Takayama K, Wang C, Besra GS (2005) Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin Microbiol Rev 18: 81–101.
[35]
Rhoades E, Hsu F, Torrelles JB, Turk J, Chatterjee D, et al. (2003) Identification and macrophage-activating activity of glycolipids released from intracellular Mycobacterium bovis BCG. Mol Microbiol 48: 875–888.
[36]
Leemans JC, Wieland CW, Florquin S, van der Poll T, Vervoordeldonk MJ (2006) Mice overexpressing p40 in lungs have reduced leucocyte influx and slightly impaired resistance during tuberculosis. Immunology 117: 409–418.
[37]
Olleros ML, Vesin D, Martinez-Soria E, Allenbach C, Tacchini-Cottier F, et al. (2007) Interleukin-12p40 overexpression promotes interleukin-12p70 and interleukin-23 formation but does not affect bacille Calmette-Guerin and Mycobacterium tuberculosis clearance. Immunology 122: 350–361.
[38]
Rao V, Gao F, Chen B, Jacobs WR Jr, Glickman MS (2006) Trans -cyclopropanation of mycolic acids on trehalose dimycolate suppresses Mycobacterium tuberculosis -induced inflammation and virulence. J Clin Invest 116: 1660–1667.
[39]
Rao V, Fujiwara N, Porcelli SA, Glickman MS (2005) Mycobacterium tuberculosis controls host innate immune activation through cyclopropane modification of a glycolipid effector molecule. J Exp Med 201: 535–543.
[40]
Oswald IP, Dozois CM, Petit JF, Lemaire G (1997) Interleukin-12 synthesis is a required step in trehalose dimycolate-induced activation of mouse peritoneal macrophages. Infect Immun 65: 1364–1369.
[41]
Geisel RE, Sakamoto K, Russell DG, Rhoades ER (2005) In vivo activity of released cell wall lipids of Mycobacterium bovis bacillus Calmette-Guerin is due principally to trehalose mycolates. J Immunol 174: 5007–5015.
[42]
Rachman H, Strong M, Ulrichs T, Grode L, Schuchhardt J, et al. (2006) Unique transcriptome signature of Mycobacterium tuberculosis in pulmonary tuberculosis. Infect Immun 74: 1233–1242.
[43]
Yuan Y, Zhu Y, Crane DD, Barry CE 3rd (1998) The effect of oxygenated mycolic acid composition on cell wall function and macrophage growth in Mycobacterium tuberculosis. Mol Microbiol 29: 1449–1458.
[44]
Coats SR, Reife RA, Bainbridge BW, Pham TT, Darveau RP (2003) Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at toll-like receptor 4 in human endothelial cells. Infect Immun 71: 6799–6807.
[45]
Hajjar AM, Ernst RK, Tsai JH, Wilson CB, Miller SI (2002) Human Toll-like receptor 4 recognizes host-specific LPS modifications. Nat Immunol 3: 354–359.
[46]
Andersen-Nissen E, Smith KD, Strobe KL, Barrett SL, Cookson BT, et al. (2005) Evasion of Toll-like receptor 5 by flagellated bacteria. Proc Natl Acad Sci U S A 102: 9247–9252.
[47]
Pathak SK, Basu S, Bhattacharyya A, Pathak S, Kundu M, et al. (2005) Mycobacterium tuberculosis lipoarabinomannan-mediated IRAK-M induction negatively regulates Toll-like receptor-dependent interleukin-12 p40 production in macrophages. J Biol Chem 280: 42794–42800.
[48]
Reed MB, Domenech P, Manca C, Su H, Barczak AK, et al. (2004) A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature 431: 84–87.
[49]
Pathak SK, Basu S, Basu KK, Banerjee A, Pathak S, 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.
[50]
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.
[51]
Cooper AM, Khader SA (2007) IL-12p40: an inherently agonistic cytokine. Trends Immunol 28: 33–38.
[52]
Bogdan C, Schleicher U (2006) Production of interferon-gamma by myeloid cells–fact or fancy? Trends Immunol 27: 282–290.
[53]
Fenton MJ, Vermeulen MW, Kim S, Burdick M, Strieter RM, et al. (1997) Induction of gamma interferon production in human alveolar macrophages by Mycobacterium tuberculosis. Infect Immun 65: 5149–5156.
[54]
Wang J, Wakeham J, Harkness R, Xing Z (1999) Macrophages are a significant source of type 1 cytokines during mycobacterial infection. J Clin Invest 103: 1023–1029.
[55]
Murphy TL, Cleveland MG, Kulesza P, Magram J, Murphy KM (1995) Regulation of interleukin 12 p40 expression through an NF-kappa B half-site. Mol Cell Biol 15: 5258–5267.
[56]
Tullius MV, Harth G, Horwitz MA (2001) High extracellular levels of Mycobacterium tuberculosis glutamine synthetase and superoxide dismutase in actively growing cultures are due to high expression and extracellular stability rather than to a protein-specific export mechanism. Infect Immun 69: 6348–6363.
[57]
Ellingsen E, Morath S, Flo T, Schromm A, Hartung T, et al. (2002) Induction of cytokine production in human T cells and monocytes by highly purified lipoteichoic acid: involvement of Toll-like receptors and CD14. Med Sci Monit 8: BR149–156.
[58]
Grangette C, Nutten S, Palumbo E, Morath S, Hermann C, et al. (2005) Enhanced antiinflammatory capacity of a Lactobacillus plantarum mutant synthesizing modified teichoic acids. Proc Natl Acad Sci U S A 102: 10321–10326.
[59]
Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, et al. (1999) Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11: 443–451.
