Lipoarabinomannan from Mycobacterium tuberculosis Promotes Macrophage Survival by Phosphorylating Bad through a Phosphatidylinositol 3-Kinase/Akt Pathway

Abstract

Efforts in prevention and control of tuberculosis suffer from the lack of detailed knowledge of the mechanisms used by pathogenic mycobacteria for survival within host cell macrophages. The exploitation of host cell signaling pathways to the benefit of the pathogen is a phenomenon that deserves to be looked into in detail. We have tested the hypothesis that lipoarabinomannan (LAM) from the virulent species of Mycobacterium tuberculosis possesses the ability to modulate signaling pathways linked to cell survival. The Bcl-2 family member Bad is a proapoptotic protein. Phosphorylation of Bad promotes cell survival in many cell types. We demonstrate that man-LAM stimulates Bad phosphorylation in a phosphatidylinositol 3-kinase (PI-3K)-dependent pathway in THP-1 cells. Man-LAM activated PI-3K. LAM-stimulated phosphorylation of Bad was abrogated in cells transfected with a dominant-negative mutant of PI-3K (Δp85), indicating that activation of PI-3K is sufficient to trigger phosphorylation of Bad by LAM. Since phosphorylation of Bad occurred at serine 136, the target of the serine/threonine kinase Akt, the effect of LAM on Akt kinase activity was tested. Man-LAM could activate Akt as evidenced from phosphorylation of Akt at Thr308 and by the phosphorylation of the exogenous substrate histone 2B. Akt activation was abrogated in cells transfected with Δp85. The phosphorylation of Bad by man-LAM was abrogated in cells transfected with a kinase-dead mutant of Akt. These results establish that LAM-mediated Bad phosphorylation occurs in a PI-3K/Akt-dependent manner. It is therefore the first demonstration of the ability of a mycobacterial virulence factor to up-regulate a signaling pathway involved in cell survival. This is likely to be one of a number of virulence-associated mechanisms by which bacilli control host cell apoptosis. Efforts in prevention and control of tuberculosis suffer from the lack of detailed knowledge of the mechanisms used by pathogenic mycobacteria for survival within host cell macrophages. The exploitation of host cell signaling pathways to the benefit of the pathogen is a phenomenon that deserves to be looked into in detail. We have tested the hypothesis that lipoarabinomannan (LAM) from the virulent species of Mycobacterium tuberculosis possesses the ability to modulate signaling pathways linked to cell survival. The Bcl-2 family member Bad is a proapoptotic protein. Phosphorylation of Bad promotes cell survival in many cell types. We demonstrate that man-LAM stimulates Bad phosphorylation in a phosphatidylinositol 3-kinase (PI-3K)-dependent pathway in THP-1 cells. Man-LAM activated PI-3K. LAM-stimulated phosphorylation of Bad was abrogated in cells transfected with a dominant-negative mutant of PI-3K (Δp85), indicating that activation of PI-3K is sufficient to trigger phosphorylation of Bad by LAM. Since phosphorylation of Bad occurred at serine 136, the target of the serine/threonine kinase Akt, the effect of LAM on Akt kinase activity was tested. Man-LAM could activate Akt as evidenced from phosphorylation of Akt at Thr308 and by the phosphorylation of the exogenous substrate histone 2B. Akt activation was abrogated in cells transfected with Δp85. The phosphorylation of Bad by man-LAM was abrogated in cells transfected with a kinase-dead mutant of Akt. These results establish that LAM-mediated Bad phosphorylation occurs in a PI-3K/Akt-dependent manner. It is therefore the first demonstration of the ability of a mycobacterial virulence factor to up-regulate a signaling pathway involved in cell survival. This is likely to be one of a number of virulence-associated mechanisms by which bacilli control host cell apoptosis. lipoarabinomannan phosphatidylinositol phosphatidylinositol 3-kinase Despite the potential role of the macrophage in the eradication of microbes, pathogenic Mycobacterium species have survived down the ages as some of the most successful in evading macrophage surveillance mechanisms in a manner that ensures their survival and replication inside the macrophage. A variety of mechanisms contribute to the survival of Mycobacterium tuberculosiswithin macrophages (1Fenton M.J. Vermeulen M.W. Infect. Immun. 1996; 64: 683-690Crossref PubMed Google Scholar) including inhibition of phagosome-lysosome fusion (2Meresse, S., Steele-Mortimer, O., Moreno, E., Desjardin, M., Finlay, B., and Gorvel, J.-P. (1999) Nat. Cell Biol. E183–E188Google Scholar), inhibition of the acidification of phagosomes (3Sturgill-Koszycki S. Schlesinger P.H. Chakraborty P. Haddix P.L. Collins H.L. Fok A.K. Allen R.D. Gluck S.L. Heuser J. Russell D.G. Science. 1994; 263: 678-691Crossref PubMed Scopus (1057) Google Scholar), and resistance to killing by reactive oxygen (4Jacket P.S. Andrew P.W. Lowrie D.B. Adv. Exp. Med. Biol. 1982; 155: 687-693Crossref PubMed Scopus (6) Google Scholar) and reactive nitrogen intermediates (5MacMicking J. North R.J. La Course R. Mudgett J.S. Shah S.K. Nathan C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5243-5248Crossref PubMed Scopus (902) Google Scholar, 6Chan J. Xing Y. Magliozzo R.S. Bloom B.R. J. Exp. Med. 1992; 175: 1111-1112Crossref PubMed Scopus (858) Google Scholar). M. tuberculosis produces large quantities of lipoarabinomannan (LAM).1 LAM can inhibit macrophage activation and triggering and represents a virulence factor contributing to the persistence of mycobacteria within macrophages. LAM is a complex molecule consisting of a phosphatidylinositol (PI) moiety that anchors a large mannose core to the mycobacterial cell wall (7Chatterjee D. Khoo K.-H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (297) Google Scholar, 8Hunter S.W. Brennan P.J. J. Biol. Chem. 1990; 265: 9272-9279Abstract Full Text PDF PubMed Google Scholar, 9Hunter S.W. Gaylord H. Brennan P.J. J. Biol. Chem. 1986; 261: 12345-12351Abstract Full Text PDF PubMed Google Scholar). The mannose core consists of multiple branched, arabinofuranosyl side chains. M. tuberculosis and M. leprae modify the nonreducing end of the arabinofuranosyl chains with mannose residues yielding man-LAM, whereas rapidly growing mycobacterial species have nonreducing termini of two types, the linear Ara4 and the branched Ara6 motifs, thereby giving rise to ara-LAM (7Chatterjee D. Khoo K.-H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (297) Google Scholar, 10Khoo K.-H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. J. Biol. Chem. 1996; 270: 12380-12389Abstract Full Text Full Text PDF Scopus (160) Google Scholar). LAM exhibits a wide array of immunomodulatory functions including inhibition of interferon-γ-induced functions such as macrophage microbicidal and tumoricidal activity (11Sibley L.D. Hunter S.W. Brennan P.J. Krahenbuhl J.L. Infect. Immun. 1988; 56: 1232-1236Crossref PubMed Google Scholar), scavenging of potentially cytotoxic oxygen free radicals (12Chan J. Fan X. Hunter S.W. Brennan P.J. Bloom B.R. Infect. Immun. 1991; 59: 1755-1761Crossref PubMed Google Scholar), inhibition of protein kinase C activity (13Chan, S. D., Fan, Hunter, S. W., Brennan, P. J., and Bloom, B. R. (1991)Google Scholar), and evocation of a number of cytokines such as tumor necrosis factor-α (14Barnes P.F. Chatterjee D. Brennan P.J. Rea T.H. Modlin R.L. Infect. Immun. 1992; 60: 1441-1446Crossref PubMed Google Scholar). The early response genes c-fos, KC, and JE are induced by ara-LAM but not by man-LAM (15Roach T.A. Barton C.H. Chatterjee D. Blackwell J.M. J. Immunol. 1993; 60: 1886-1896Google Scholar). The ability of man-LAM to impair responsiveness to interferon-γ and to attenuate tumor necrosis factor-α and interleukin-12 mRNA production through effects on the protein phosphatase SHP-1 has been suggested to be a major mechanism by which man-LAM promotes intracellular survival (16Knutson K.L. Hmaama Z. Herrera-Velit P. Rochford R. Reiner N.E. J. Biol. Chem. 1998; 273: 645-652Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Despite the recent advances, the intracellular signaling following interaction of the mycobacterium with host cells and the role of LAM in these processes is incompletely understood. The survival of M. tuberculosis in macrophages probably involves more than one mechanism. In this study, we have tested whether LAM plays a role in triggering a signaling pathway that suppresses the intrinsic cell death machinery of phagocytic cells. The regulation of programmed cell death, apoptosis, is an exceptionally complicated process that involves a myriad of proteins. The family of proteins that includes Bcl-2 comprises members that are both antiapoptotic and those that are proapoptotic such as Bax and Bad. Bad interacts with Bcl-2 and Bcl-XL, sequestering these proteins, and thus promotes apoptosis (17Yang E. Zha J. Jockel J. Boise L.H. Thompson C.B. Korsmeyer S.J. Cell. 1995; 80: 285-291Abstract Full Text PDF PubMed Scopus (1888) Google Scholar). Phosphorylation of Bad at either of two sites, serine residues 112 and 136 (numbering based on the sequence of murine Bad) creates consensus sites for interaction with the 14-3-3 protein. Bad is then bound to 14-3-3 instead of Bcl-2 or Bcl-XL, resulting in the liberation of the antiapoptotic proteins and promotion of cell survival. Interleukin-3 (18Yao R. Cooper G.N. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1290) Google Scholar) and other survival factors promote cell survival through their ability to stimulate phosphatidylinositol 3-kinase (PI-3K) (19Yao R. Minshall C. Arkins S. Freund G.G. Kelley K.W. J. Immunol. 1996; 156: 939-947PubMed Google Scholar) and inactivate the apoptotic factor Bad (20Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2246) Google Scholar, 21Songyang Z. Baltimore D. Cantley L. Kaplan D.R. Franke T.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11345-11350Crossref PubMed Scopus (322) Google Scholar). The PI-3K-sensitive pathway involves the activation of the serine/threonine kinase Akt or protein kinase B (22Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2215) Google Scholar, 23Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (965) Google Scholar, 24Kaufmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1069) Google Scholar, 25Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1182) Google Scholar) and direct phosphorylation of Bad at serine 136 (26Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Goto, Y., and Nunez, G. (1997) 91, 231–241Google Scholar, 27del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1981) Google Scholar, 28Eves E.M. Xiong W. Bellacosa A. Kennedy S.G. Tsichlis P.N. Rosner M.R. Hay N. Mol. Cell. Biol. 1998; 18: 2143-2152Crossref PubMed Scopus (175) Google Scholar, 29Blume-Jensen P. Janknecht R. Hunter T. Curr. Biol. 1998; 8: 779-782Abstract Full Text Full Text PDF PubMed Google Scholar). Akt/protein kinase B is the major downstream target of receptor tyrosine kinases that signal via the PI-3K. Activated protein kinase B has been implicated in glucose metabolism, transcriptional control, and the regulation of apoptosis in many cell types (30Galetic I. Andjelkovic M. Meier R. Brodbeck D. Park J. Hemmings B.A. Pharmacol. Ther. 1999; 82: 409-425Crossref PubMed Scopus (95) Google Scholar, 31Datta S. Brunet A. Greenberg M. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3711) Google Scholar). In addition to Bad, Akt also phosphorylates caspase 9 (32Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2722) Google Scholar), forkhead transcription factor (33Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5380) Google Scholar, 34Kops G.J.P.L. de Ruiter N.D. Vries-Smits A.M.M. Powell D.R. Bos J.L. Burgering B.M.T. Nature. 1999; 398: 630-634Crossref PubMed Scopus (949) Google Scholar), and the IκB kinase, thereby activating NF-κB (35Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1883) Google Scholar, 36Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1664) Google Scholar). In addition to phosphorylation at serine 136 mediated by protein kinase B, Bad undergoes protein kinase A- (37Harada H. Becknell B. Wilm M. Mann M. Huang L.J. Taylor S.S. Scott J.D. Korsmeyer S.J. Mol. Cell. 1999; 3: 413-422Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar) and p90RSK- (38Tan Y. Ruan H. Demeer M.R. Comb M.J. J. Biol. Chem. 1999; 274: 34859-34867Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) mediated phosphorylation on serine 112. The Ca2+-activated protein phosphatase calcineurin can dephosphorylate Bad, reversing the phosphorylation at both serine 112 and serine 136 (39Wang H.-G. Pathan N. Ethell I. Krajewski S. Yamaguchi Y. Shibasaki F. McKeon F. Bobo T. Franke T.F. Reed J.C. Science. 1999; 284: 339-343Crossref PubMed Scopus (960) Google Scholar). A third phosphorylation site at serine 155 has recently been identified (40Lizcano J.M. Morrice N. Cohen P. Biochem. J. 2000; 349: 547-557Crossref PubMed Scopus (256) Google Scholar, 41Zhou X.-M. Liu Y. Payne G. Lutz R.J. Chittenden T. J. Biol. Chem. 2000; 275: 25046-25051Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 42Tan Y. Demeter M.R. Ruan H. Comb M.J. J. Biol. Chem. 2000; 275: 25865-25869Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 43Datta S.R. Katsov A. Hu L. Petros A. Fesik S.W. Yaffe M.B. Greenberg M.E. Mol. Cell. 2000; 6: 41-51Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar). When Bad is bound to prosurvival Bcl-2 family members, Bad serine 155 phosphorylation requires the prior phosphorylation at serine 136, which recruits 14-3-3 proteins that then function to increase the accessibility of serine 155 to survival-promoting kinases (43Datta S.R. Katsov A. Hu L. Petros A. Fesik S.W. Yaffe M.B. Greenberg M.E. Mol. Cell. 2000; 6: 41-51Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar). Bad is not a ubiquitously expressed protein. Nevertheless, several major signaling pathways influence cell survival through their effects on the phosphorylation state of Bad (44Downward J. Nat. Cell Biol. 1999; 1: E33-E35Crossref PubMed Scopus (203) Google Scholar). We demonstrate that man-LAM from the virulent Erdman strain of M. tuberculosis promotes phosphorylation of Bad at serine 136 through a PI-3K/Akt pathway in the human cell line THP-1 and hypothesize that this probably represents one of the mechanisms by which man-LAM promotes cell survival to the benefit of the pathogen. Histone 2B was purchased from Roche Molecular Biochemicals. Phorbol 12-myristate 13-acetate was purchased from Sigma. Protein A/G Plus-agarose, rabbit anti-p85 phosphoinositide 3-kinase, goat anti-Akt, and rabbit anti-Bad antibody were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phophotyrosine antibody was from Life Technologies, Inc. Wortmannin, LY294002, PD98059, and SB203580 were from Calbiochem. Phosphorylation Bad and and Akt and the were from Inc. other were from Sigma. THP-1 cells from a with are cells from the These were from the for Cell and were from Life Technologies, Inc. The cell line was in and The cells were at and in a THP-1 cells were with 12-myristate 13-acetate to of the to a The cells were with and to a of Cell was to be by the were in of were of by in for with LAM. were on THP-1 cells in were transfected with of or the in with to the The dominant-negative mutant of was in the of (Δp85), which to the of The mutant of Akt the man-LAM and ara-LAM were by through of Man-LAM was from the virulent Erdman strain of M. LAM was at a of in and with for Akt protein kinase cells were in Franke T.F. A. Yang Kaplan D.R. Tsichlis P.N. Mol. Cell. Biol. 1995; PubMed Scopus Google were with and kinase were with the histone 2B as substrate Franke T.F. A. Yang Kaplan D.R. Tsichlis P.N. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). at for the was by protein and the was by by of cell with anti-p85 PI-3K were and PI-3K were M. Kaplan D. T. Cantley L. Biochem. J. PubMed Scopus Google Scholar). were for at were and on Y. H. Mol. Cell. Biol. PubMed Scopus Google Scholar), and was by or with LAM were in A and for and for at the and were by at for The were and in A. Cell were as and anti-Akt, or the The were on for Akt and for Bad) and to were with with antibody at by goat antibody to for at and the of THP-1 cells with man-LAM, the phosphorylation of Bad was Man-LAM from the virulent Erdman species of M. tuberculosis the phosphorylation of Bad at in a manner LAM from a growing species not an effect to that of man-LAM not Phosphorylation of Bad at serine 112 was not The phosphorylation of Bad at serine 136 occurs through a PI-3K/Akt-dependent pathway (26Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Goto, Y., and Nunez, G. (1997) 91, 231–241Google Scholar, 27del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1981) Google Scholar, 28Eves E.M. Xiong W. Bellacosa A. Kennedy S.G. Tsichlis P.N. Rosner M.R. Hay N. Mol. Cell. Biol. 1998; 18: 2143-2152Crossref PubMed Scopus (175) Google Scholar, 29Blume-Jensen P. Janknecht R. Hunter T. Curr. Biol. 1998; 8: 779-782Abstract Full Text Full Text PDF PubMed Google Scholar). were with prior to with man-LAM in to their ability to Bad The PI-3K and Bad phosphorylation at serine 136 in a manner The and not influence Bad phosphorylation not It was that man-LAM through PI-3K to Bad. Since Bad phosphorylation was abrogated by PI-3K in PI-3K activity was tested with Man-LAM was to stimulate PI-3K activity in a manner. that of PI-3K were used to kinase of PI-3K is by to the through interaction of in with proteins. with man-LAM a increase in the of the in the indicating from the phosphorylation of mechanism of PI-3K activation D.R. M. B. M. Cantley L. Cell. Full Text PDF PubMed Scopus Google Scholar). Cell were with anti-p85 and by with man-LAM the tyrosine phosphorylation of the of PI-3K in a manner The PI-3K-sensitive pathway of Bad phosphorylation involves the activation of the protein kinase Akt protein kinase and the phosphorylation of Bad at serine The interaction of the of Akt with the of PI-3K a in Akt, a more substrate for the kinase D.R. M. A. Morrice N. D.G. P. C.B. D. A. M. Curr. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, D.R. S.R. C.B. Cohen P. Curr. Biol. 1997; Full Text Full Text PDF PubMed Google Scholar, D. T. C.B. F. Science. 1997; PubMed Scopus Google Scholar). Akt phosphorylation on Thr308 by kinase and on is for whether Akt is a downstream of PI-3K signaling in THP-1 Akt phosphorylation on Thr308 was with Thr308 by of the phosphorylation of Akt at Thr308 in a and manner. LAM histone 2B phosphorylation in a manner indicating of Akt kinase Histone phosphorylation by man-LAM was by the PI-3K and man-LAM, of cells to ara-LAM for not in of Akt kinase activity establish whether Akt is the serine/threonine kinase that is downstream of PI-3K in the man-LAM signaling the effect of of the mutant of on Akt kinase activation was Thr308 phosphorylation of Akt was abrogated by of the mutant of PI-3K but not by the control In with Bad phosphorylation at serine 136 was also abrogated in cells not In to establish a Akt kinase activation and the phosphorylation of Bad at serine 136 induced by we transfected THP-1 cells with a kinase-dead mutant of Akt and the phosphorylation of Bad. abrogated the phosphorylation of Bad following by whereas control such effect The of the of M. tuberculosis is M. tuberculosis is a intracellular pathogen. the function of macrophages is to and mycobacteria have to the mechanisms of macrophages. to the ability of M. tuberculosis to the host and or is the of the to the host and and within macrophages. apoptosis to host M. tuberculosis macrophages apoptosis in response to M. J. M.J. H. Infect. Immun. 1997; PubMed Google Scholar). recently has been that control of host cell apoptosis is a virulence-associated of M. with virulent the ability to apoptosis of macrophages J. H. J. Immunol. 2000; PubMed Scopus Google Scholar). In of these the virulence factors that modulate host cell apoptosis is We to whether man-LAM from a virulent strain of M. tuberculosis could modulate cell signaling pathways to control cell survival. The phosphorylation of Bad is one of the mechanisms of of cells from programmed cell We have tested the hypothesis that man-LAM from the virulent species of M. cells from apoptosis at through phosphorylation of Bad. Man-LAM was to stimulate Bad phosphorylation on serine 136 in THP-1 cells. Bad phosphorylation at serine 136 is to via a PI-3K/Akt signaling with the PI-3K and the LAM-stimulated phosphorylation of Bad. of PI-3K was of cells with LAM. LAM to to the a phenomenon with kinase activity of this phosphorylation of mechanism of PI-3K LAM tyrosine phosphorylation of The that PI-3K activation is sufficient for Bad phosphorylation of cells with LAM was by the that of THP-1 cells with the mutant of PI-3K abrogated LAM-mediated Bad of the major functions of Akt is of cells from programmed cell This has been for several cell types including cells G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (965) Google Scholar), A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1069) Google Scholar), and cells K.L. M.J. Klippel A. J. Cell Biol. 1997; PubMed Scopus Google Scholar). This is at in to the ability of Akt to Bad at serine We to establish a Akt, and the phosphorylation of Bad. Akt phosphorylation at Thr308 was by LAM in a PI-3K-sensitive manner. Akt kinase activity by histone 2B as exogenous substrate was also by LAM. of THP-1 with the mutant of PI-3K the LAM-stimulated phosphorylation of Akt at Thr308 as as the ability of Akt to histone that Akt is a downstream of LAM-mediated PI-3K The Akt and Bad was by cells with a mutant of Akt and the phosphorylation of Bad with LAM. LAM-mediated Bad phosphorylation was in cells transfected with In we demonstrate that man-LAM from the virulent Erdman strain of M. tuberculosis Akt in a PI-3K-sensitive to the phosphorylation of Bad at serine 136, that this be one of the mechanisms by which LAM promotes macrophage cell survival. of macrophage apoptosis the mycobacteria to from into apoptotic of bacilli in this is suggested to in more microbicidal C. C. C. J. Immunol. 1997; Google Scholar). This is the first demonstration of a mycobacterial virulence factor the of a macrophage survival signaling thereby an for the survival of the pathogen. We are to and for the of the mutant of PI-3K and