From the Department of Chemistry, Bose Institute, 93/1 Acharya
Prafulla Chandra Road, Calcutta 700 009, India
Received for publication, March 29, 2000, and in revised form, September 12, 2000
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 (
p85), 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
p85. 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.
 |
INTRODUCTION |
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 tuberculosis
within macrophages (1) including inhibition of phagosome-lysosome fusion (2), inhibition of the acidification of phagosomes (3), and
resistance to killing by reactive oxygen (4) and reactive nitrogen
intermediates (5, 6). 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
(7-9). 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 (7, 10). LAM exhibits a wide array of
immunomodulatory functions including inhibition of
interferon-
-induced functions such as macrophage microbicidal and
tumoricidal activity (11), scavenging of potentially cytotoxic oxygen
free radicals (12), inhibition of protein kinase C activity (13), and
evocation of a number of cytokines such as tumor necrosis factor-
(14). The early response genes c-fos, KC, and
JE are induced by ara-LAM but not by man-LAM (15). The
ability of man-LAM to impair responsiveness to interferon-
and to
attenuate tumor necrosis factor-
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 (16).
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 (17). 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 (18) and other
survival factors promote cell survival through their ability to
stimulate phosphatidylinositol 3-kinase (PI-3K) (19) and inactivate the
apoptotic factor Bad (20, 21). The PI-3K-sensitive pathway involves the
activation of the serine/threonine kinase Akt or protein kinase B
(22-25) and direct phosphorylation of Bad at serine 136 (26-29).
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 (30, 31). In addition to
Bad, Akt also phosphorylates caspase 9 (32), forkhead transcription
factor (33, 34), and the I
B kinase, thereby activating NF-
B
(35, 36).
In addition to phosphorylation at serine 136 mediated by protein kinase
B, Bad undergoes protein kinase A- (37) and p90RSK- (38)
mediated phosphorylation on serine 112. The Ca2+-activated
protein phosphatase calcineurin can dephosphorylate Bad, reversing the
phosphorylation at both serine 112 and serine 136 (39). A third
phosphorylation site at serine 155 has recently been identified
(40-43). 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 (43). Bad is
not a ubiquitously expressed protein. Nevertheless, several major
signaling pathways influence cell survival through their effects on the
phosphorylation state of Bad (44). 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.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
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 state-specific
antibodies Bad (Ser112) and (Ser136) and Akt
(Thr308) antibodies and the Phototope-HRP Chemiluminescent
Western Detection Kit were from New England Biolabs, Inc. (Beverly,
MA). All other chemicals were from Sigma.
Cell Culture and Transfection--
THP-1 cells (derived from a
patient with acute monocytic leukemia) are mature cells from the
monocyte/macrophage lineage. These were obtained from the National
Center for Cell Science (Pune, India). Media and supplements were
obtained from Life Technologies, Inc. The cell line was maintained in
RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM
glutamine, and 20 mM sodium bicarbonate. The cells were
incubated at 5% CO2 and 95% humidity in a 37 °C chamber. THP-1 cells were treated with phorbol 12-myristate 13-acetate to induce maturation of the monocytes to a macrophage-like, adherent phenotype. The cells were washed three times with culture medium without fetal bovine serum and resuspended to a concentration of 2 × 106 cells/ml. Cell viability was determined to be >95%
by the trypan blue dye exclusion method. 2 × 106
cells were plated in each well of six-well plates. Cells were deprived
of serum by culturing in RPMI without fetal bovine serum for 12-16 h,
before treatment with LAM. Transfections were carried out on adherent
THP-1 cells (2 × 106 cells/well in six-well plates).
Cells were transfected with 2 µg of plasmid (recombinants or empty
vectors) using the Effectene Reagent (Qiagen) in RPMI with 10% fetal
bovine serum according to the manufacturer's instructions. The
dominant-negative mutant of p85 was deleted in the inter-SH2 region of
wild type p85
(
p85), which abolishes binding to the p110 subunit
of PI 3-kinase. The kinase-deficient mutant of Akt (Akt.KD) carried the
mutation K179M.
Mycobacterial Lipids--
Endotoxin-free man-LAM and ara-LAM
were kindly provided by Dr. John Belisle (Colorado State University,
Ft. Collins, CO, through NIAID, National Institutes of Health, Contract
NO1-AI-75320). Man-LAM was derived from the virulent Erdman strain
of M. tuberculosis. LAM was dissolved at a concentration of
1 mg/ml in pyrogen-free water and diluted with medium before each experiment.
Immunocomplex Akt Kinase Assay--
To assay for Akt protein
kinase activity, cells were lysed in lysis buffer (45); lysates were
incubated with anti-Akt antibody, and kinase assays were carried out
with the immunoprecipitates using histone 2B as substrate (45). After
incubation at room temperature for 30 min, the reaction was stopped by
adding protein gel denaturing buffer, and the mixture was separated by
SDS-polyacrylamide gel electrophoresis, followed by autoradiography.
Immunocomplex PI 3-kinase Assay--
Following
immunoprecipitation of cell lysates with anti-p85 PI-3K antibody,
immunoprecipitates were washed, and PI-3K assays were performed (46).
Reactions were carried out for 15 min at room temperature,
phosphorylated lipid products were extracted and separated on TLC
plates (47), and incorporated radioactivity was measured by liquid
scintillation counting.
Preparation of Cell Membranes--
Cells after treatment without
or with LAM were washed, resuspended in buffer A (137 mM
NaCl, 2.7 mM KCl, 8.1 mM
Na2HPO4, 1.5 mM
KH2PO4, 2.5 mM EDTA, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM pefabloc), sonicated twice for 10 s each, and
centrifuged for 5 min at 800 × g. After discarding the
nuclei and unbroken cells, membranes were prepared by
ultracentrifugation at 250,000 × g for 60 min. The
pellets (membranes) were washed and suspended in buffer A.
Immunoblotting--
Cell lysates were prepared as described
above and immunoprecipitated using anti-Akt, anti-Bad, or the
respective phosphospecific antibodies. The immunoprecipitates were
fractionated on SDS-polyacrylamide (10% for Akt and 12% for Bad) gels
and transferred to nitrocellulose membranes. Membranes were blocked
with 5% nonfat dry milk, incubated with primary antibody overnight at
4 °C, followed by goat anti-rabbit secondary antibody conjugated to
horseradish peroxidase for 1 h at room temperature, and finally
visualized using the Phototope-HRP Western detection kit.
 |
RESULTS |
Phosphorylation of Bad--
Following treatment of THP-1 cells
with man-LAM, the phosphorylation status of Bad was examined using
phosphospecific antibodies. Man-LAM from the virulent Erdman species of
M. tuberculosis promoted the phosphorylation of Bad at
Ser136 in a time-dependent manner (Fig.
1A). LAM from a fast growing species (ara-LAM) did not exhibit an effect equivalent to that of
man-LAM (data not shown). Phosphorylation of Bad at serine 112 was not
observed.

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Fig. 1.
Man-LAM-induced phosphorylation of Bad in
THP-1 cells. Differentiated THP-1 cells in serum-free
medium were incubated with man-LAM (2 µg/ml) for different
periods of time, followed by immunoprecipitation of lysates with
anti-Bad antibody, SDS-polyacrylamide gel electrophoresis, and
immunoblotting and development by enhanced chemiluminescence as
described under "Experimental Procedures." A, a
representative Western blot using anti-phospho-Bad Ser136.
Total Bad was immunoblotted to confirm equal loading. B,
cells were treated with either wortmannin (lanes
e, f, and g, representing
concentrations of 1.5, 15, and 150 nM, respectively) for 45 min or LY294002 (lanes h, i, and
j, representing concentrations of 2, 20, and 200 µM, respectively) for 4 h or vehicle
(DMSO) (lanes a and c,
representing treatment for 45 min, and lanes b
and d, representing treatment for 4 h, respectively).
Cells were then either not treated (lanes a and
b) or treated (lanes c-j) with 2 µg/ml man-LAM for 60 min. Bad phosphorylation was then visualized
using phospho-Bad antibodies after lysing the cells. Total Bad was
immunoblotted to confirm equal loading.
|
|
PI-3K Inhibitors Block Bad Phosphorylation--
The
phosphorylation of Bad at serine 136 occurs through a
PI-3K/Akt-dependent pathway (26-29). Cells were pretreated
with selective inhibitors prior to stimulation with man-LAM in order to
test their ability to block Bad phosphorylation. The PI-3K inhibitors wortmannin and LY294002 inhibited Bad phosphorylation at
serine 136 in a dose-dependent manner (Fig.
1B). The MEKK1 inhibitors PD90859 and SB20358
did not influence Bad phosphorylation (data not shown). It was inferred
that man-LAM signals through PI-3K to phosphorylate Bad.
LAM Activates PI-3K--
Since man-LAM-induced Bad phosphorylation
was abrogated by PI-3K inhibitors, in vitro PI-3K activity
was tested after treatment with man-LAM. Man-LAM was found to stimulate
PI-3K activity (Fig. 2A) in a
time-dependent manner. Control experiments showed that equal amounts of precipitated PI-3K were used to assay kinase activity.

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Fig. 2.
Man-LAM-induced activation of PI 3-kinase in
THP-1 cells. A, serum-starved cells were exposed to
man-LAM(2 µg/ml) for different time periods followed by
immunoprecipitation of lysates with anti-p85 PI 3-kinase antibody. PI
3-kinase activity was assayed using PI as substrate as described under
"Experimental Procedures," and PIP was detected by autoradiography
after separation by TLC (top panel). The
middle panel represents a densitometric analysis
of the autoradiograms. Data represent the mean ± S.E. of three
independent experiments. The bottom panel is a
Western blot of anti-p85 in the immunoprecipitate. Lanes
a, b, c, d, and
e represent the time points 0, 5, 10, 30, and 60 min,
respectively. B, translocation of PI-3K to the membrane. The
top panel shows the effect of man-LAM (2 µg/ml)
treatment for different time periods on the translocation of p85 to
the membrane fraction as measured by Western blotting using anti-p85
antibody. Lanes a, b, c,
and d represent time points 0, 10, 30, and 60 min,
respectively. The bottom panel is a densitometric
analysis of the blot. Error bars illustrate S.E.
from three independent experiments. C, tyrosine
phosphorylation of PI-3K by LAM. Cells after treatment were lysed and
immunoprecipitated with anti-p85 , followed by immunoblotting with
anti-phosphotyrosine antibody as described under "Experimental
Procedures." The top panel shows the effect of
man-LAM (2 µg/ml) treatment for different time periods on the
tyrosine phosphorylation of p85 . The bottom
panel is a representative Western blot of anti-p85 to
show that the same amount of p85 was present in each sample.
Lanes a, b, c, and
d represent the time points 0, 10, 30, and 60 min.
|
|
Activation of heterodimeric (p85-p110) PI-3K is often promoted by
recruitment to the plasma membrane through interaction of SH2 domains
in p85 with tyrosine-phosphorylated proteins. Treatment with man-LAM
caused a time-dependent increase in the amount of the p85
subunit detected in the membrane fraction, indicating translocation
from the cytosol (Fig. 2B). Tyrosine phosphorylation of p85
might represent another mechanism of PI-3K activation (48). Cell
lysates were immunoprecipitated with anti-p85 and analyzed by Western
blotting with anti-phosphotyrosine antibody. man-LAM treatment
increased the tyrosine phosphorylation of the p85
subunit of PI-3K
in a time-dependent manner (Fig. 2C).
Akt Activation by LAM--
The PI-3K-sensitive pathway of Bad
phosphorylation involves the activation of the protein kinase Akt (or
protein kinase B) and the phosphorylation of Bad at serine 136. The
interaction of the amino-terminal pleckstrin homology domain of
Akt with the phospholipid product of PI-3K induces a conformational
change in Akt, making it a more efficient substrate for the
phosphatidylinositide-dependent kinase 1 (49-51).
Stimulus-induced Akt phosphorylation on Thr308 by
phosphatidylinositide-dependent kinase 1 and on
Ser473 is required for maximal activity. To determine
whether Akt is a downstream effector of man-LAM-induced PI-3K signaling
in THP-1 cells, Akt phosphorylation on Thr308 was measured
using immunoblotting with phosphospecific Thr308 anti-Akt
antibody, followed by densitometric analysis of the autoradiograms.
Man-LAM-stimulated phosphorylation of Akt at Thr308 in a
dose-dependent (Fig.
3A) and
time-dependent (Fig. 3B) manner.

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Fig. 3.
Effect of man-LAM on Akt and Bad
phosphorylation. A, serum-starved cells were exposed to
man-LAM at different concentrations for 30 min. Lysates were
immunoprecipitated with anti-Akt antibody followed by
immunoprecipitation with phospho-Akt Thr308 antibody.
Immunoprecipitates were separated on SDS-polyacrylamide gels, followed
by Western blotting with phospho-Akt Thr308, development by
enhanced chemiluminescence, and densitometric analysis (top
panel). Data represent the mean ± S.E. of three
independent experiments. The bottom panel is a
representative Western blot showing that equal amounts of
immunoprecipitated Akt protein were detected by immunoblotting in each
sample. B, cells were exposed to man-LAM for different time
periods. Lysates were immunoprecipitated with anti-Akt antibody
followed by Western blotting with control Akt or phospho-Akt
Thr308. The bottom panel is a
densitometric analysis of the autoradiograms. Error
bars represent S.E. of three independent determinations.
C, cells were treated with man-LAM for different time
periods, and Akt kinase activity was measured following
immunoprecipitation with anti-Akt antibody using histone 2B as
substrate (top panel). The bottom
panel shows the amount of immunoprecipitated Akt protein in
each sample. D, cells were treated with either 100 nM wortmannin (lane c) for 45 min or
50 µM LY294002 (lane f) for 4 h or vehicle (DMSO) (lanes a and
b representing treatment for 45 min and lanes
d and e representing treatment for 4 h,
respectively). Cells were then either not treated (lanes
a and d) or treated (lanes
b, c, e, and f) with 2 µg/ml man-LAM for 60 min. Cells were treated either with medium
(lane g) or with 2 µg/ml man-LAM
(lane h) or with 2 µg/ml ara-LAM
(lane i) for 60 min. Akt kinase activity was
measured using histone 2B as substrate. E, cells were
transfected with a dominant negative mutant of p85 (lanes
c and d) or the control empty vector
(lanes a and b) using Effectene as
described. After transfection, cells were serum-starved and stimulated
without (lanes a and c) or with
(lanes b and d) man-LAM (2 µg/ml)
for 60 min. Akt phosphorylation was then visualized (lower
blot) after lysing the cells. The upper
blot shows that the same amount of Akt protein was present
in each sample. F, cells were transfected with a
kinase-deficient mutant of Akt (Akt.KD) (lanes c
and d) or with control empty vector (lanes
a and b). After transfection, cells were
serum-starved and stimulated without (lanes a and
c) or with (lanes b and d)
man-LAM (2 µg/ml) for 60 min. Bad phosphorylation was then visualized
(lower blot) after lysing the cells. The
upper blot shows that the same amount of Bad
protein was present in each sample.
|
|
Stimulation of Akt Kinase Activity by Man-LAM--
LAM stimulated
histone 2B phosphorylation in a time-dependent manner (Fig.
3C), indicating LAM-induced stimulation of Akt kinase
activity. Histone phosphorylation stimulated by man-LAM was inhibited
by the PI-3K inhibitors wortmannin and LY294002 (Fig. 3D).
Unlike man-LAM, exposure of cells to ara-LAM for 60 min did not result
in stimulation of Akt kinase activity (Fig. 3D,
lanes g-i).
Expression of a Dominant Negative Mutant of p85
Abolishes Akt
Kinase Activation--
To establish whether Akt is the
serine/threonine kinase that is downstream of PI-3K in the man-LAM
signaling pathway, the effect of expression of the dominant negative
mutant of p85
on LAM-induced Akt kinase activation was assessed.
Thr308 phosphorylation of Akt was abrogated by expression
of the
p85 mutant of PI-3K but not by the control empty vector (Fig.
3E). In harmony with this, Bad phosphorylation at serine 136 was also abrogated in cells expressing
p85 (data not shown).
Kinase-deficient Akt Mutant (Akt.KD) Blocks Man-LAM-induced
Phosphorylation of Bad--
In order to establish a connection between
Akt kinase activation and the phosphorylation of Bad at serine 136 induced by LAM, we transfected THP-1 cells with a kinase-dead mutant
(K179M) of Akt and assessed the phosphorylation status of Bad. Akt.KD
abrogated the phosphorylation of Bad following stimulation by LAM,
whereas control empty vector had no such effect (Fig.
3F).
 |
DISCUSSION |
The molecular basis of the pathogenicity of M. tuberculosis is poorly understood. M. tuberculosis is a
facultative intracellular pathogen. Whereas the normal function of
macrophages is to engulf and destroy microorganisms, mycobacteria have
evolved ways to circumvent the defense mechanisms of macrophages.
Central to the ability of M. tuberculosis to infect the host
and cause active or latent disease is the propensity of the tubercle
bacillus to enter the host mononuclear phagocyte and survive and
multiply within macrophages. Macrophage apoptosis contributes to host
defense against M. tuberculosis infection. Human alveolar
macrophages undergo apoptosis in response to M. tuberculosis
infection (52). Very recently it has been demonstrated that bacillary
control of host cell apoptosis is a virulence-associated phenotype of M. tuberculosis, with virulent strains having the ability to
evade apoptosis of infected macrophages (53). In light of these
observations, understanding the virulence factors that may modulate
host cell apoptosis is necessary. We chose to study whether
man-LAM from a virulent strain of M. tuberculosis could
modulate cell signaling pathways known to control cell survival.
The phosphorylation of Bad is one of the mechanisms of protection of
cells from programmed cell death. We have tested the hypothesis that
man-LAM from the virulent species of M. tuberculosis protects cells from apoptosis at least partly through phosphorylation of Bad. Man-LAM was found to stimulate Bad phosphorylation on serine
136 in THP-1 cells. Bad phosphorylation at serine 136 is believed to
occur via a PI-3K/Akt signaling pathway. Consistent with this, the
PI-3K inhibitors wortmannin and LY294002 inhibited the LAM-stimulated
phosphorylation of Bad. Activation of PI-3K was observed after
stimulation of cells with LAM. Furthermore, LAM caused p85 to
translocate to the membrane, a phenomenon associated with enhanced
lipid kinase activity of this enzyme. Tyrosine phosphorylation of p85
may represent another mechanism of PI-3K activation. LAM stimulated
tyrosine phosphorylation of p85. The fact that PI-3K activation is
sufficient for Bad phosphorylation upon treatment of cells with LAM was
demonstrated by the fact that transfection of THP-1 cells with the
dominant negative mutant (
p85) of PI-3K abrogated LAM-mediated Bad
phosphorylation. One of the major functions of Akt is protection of
cells from programmed cell death. This protection has been demonstrated
for several cell types including COS cells (23), fibroblasts (24), and
neuronal cells (55). This is at least in part due to the ability of Akt
to phosphorylate Bad at serine 136. We attempted to establish a
connection between PI-3K, Akt, and the phosphorylation of Bad. Akt
phosphorylation at Thr308 was stimulated by LAM in a
PI-3K-sensitive manner. Akt kinase activity assessed by using histone
2B as exogenous substrate was also stimulated by LAM. Transfection of
THP-1 with the
p85
mutant of PI-3K abolished the LAM-stimulated
phosphorylation of Akt at Thr308 as well as the ability of
Akt to phosphorylate histone 2B, establishing that Akt is a downstream
effector of LAM-mediated PI-3K signaling. The connection between Akt
and Bad was similarly established by transfecting cells with a
kinase-deficient mutant of Akt (Akt.KD) and assessing the
phosphorylation of Bad after stimulation with LAM. LAM-mediated Bad
phosphorylation was abolished in cells transfected with Akt.KD. In
summary, we demonstrate that man-LAM from the virulent Erdman strain of
M. tuberculosis activates Akt in a PI-3K-sensitive manner,
leading to the phosphorylation of Bad at serine 136, suggesting that
this may be one of the mechanisms by which LAM directly promotes macrophage cell survival. Inhibition of macrophage apoptosis would allow the mycobacteria to escape from being packaged into apoptotic bodies. Uptake of bacilli packaged in this way is suggested to result
in more effective microbicidal processing (54).
This is the first demonstration of a mycobacterial virulence factor
having the capability of up-regulating a macrophage survival signaling
pathway, thereby creating an environment favorable for the survival of
the pathogen.
We are grateful to Drs. Robert Farase and
Kenneth Walsh for the gifts of the
p85 mutant of PI-3K and Akt.KD, respectively.
Published, JBC Papers in Press, October 3, 2000, DOI 10.1074/jbc.M002650200
The abbreviations used are:
LAM, lipoarabinomannan;
PI, phosphatidylinositol;
PI-3K, phosphatidylinositol 3-kinase.
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