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,
*
Department of Medicine,
Inflammation Program, and
Graduate Program in Immunology, University of Iowa and Veterans Affairs Medical Center, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)3 are
central to all stages of tuberculosis (4, 5), but the
molecular mechanisms of antituberculous activity by human M are
incompletely understood. We have used an in vitro model in which
extracellular ATP (ATPe) induces the killing of
virulent M. tuberculosis within human M to characterize
the biochemical pathways that regulate mycobactericidal activity
(6). This approach is based on the demonstration by Kaplan
and colleagues (7) that ATPe
stimulates M killing of the attenuated vaccine strain M.
bovis bacille Calmette-Guérin (BCG) and on studies by Lammas
et al. (8) indicating the involvement of
P2X7 cell surface
receptors for ATP in bactericidal activity toward BCG. We have recently
demonstrated that ATPe stimulates killing of
virulent M. tuberculosis in human M and that
P2X7-dependent activation
of host phospholipase D (PLD) is tightly coupled to this response
(6).
In this study, we present further characterization of the biochemical
mechanisms that regulate ATPe-induced
antimycobacterial activity. We hypothesized that increases in M
cytosolic Ca2+
[Ca2+]c would be required
based on 1) our recent demonstration that pharmacologic elevation of
[Ca2+]c inhibits the
intracellular viability of M. tuberculosis during initial
infection of human M (9); 2) evidence that
[Ca2+]c is a key
regulator of PLD activity in phagocytic leukocytes
(10, 11, 12); and 3) the fact that ligation of M
P2X7 receptors produces a
significant rise in
[Ca2+]c due to influx
from the extracellular space via both the rapid opening of a
cation-selective channel as well as the subsequent production of a
large nonselective membrane pore permeable to molecules 
900 Da
(13, 14, 15, 16). Although Lammas et al. (8) reported
that Ca2+ is not required for
ATPe-induced killing of intracellular BCG, we
reasoned that bactericidal activity toward virulent M.
tuberculosis might exhibit distinct biochemical requirements.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unless noted, all materials were from previously published
sources (6, 9). The H37Rv and Erdman strains of M.
tuberculosis were obtained from the American Tissue Type Culture
Collection (Manassas, VA). The CDC1551 M. tuberculosis
strain was generously provided by Dr. T. Shinnick (Centers for Disease
Control and Prevention, Atlanta, GA). Virulent strains of M.
tuberculosis (H37Rv, Erdman, and CDC1551) were cultured and
prepared for use in experiments as noted previously (17).
Briefly, aliquots of frozen M. tuberculosis stocks in 7H9
broth were thawed, cultured for 9 days on 7H11 agar at 37°C in 5%
CO2-95% air, scraped from agar plates, and
suspended in RPMI 1640 by vortexing briefly. After settling, the
supernatant was transferred to a new tube and allowed to settle once
again. An aliquot of this final M. tuberculosis suspension
was counted in a Petroff-Hauser chamber, and the concentration of
bacteria was adjusted for use in experiments. M.
tuberculosis preparations contained 
90% single bacteria, with
80% viability by determination of CFUs (17). The
effects of various experimental manipulations on the viability of
M. tuberculosis were determined by analysis of
CFUs.
Preparation of M
PBMC were isolated from healthy, purified protein
derivative-negative adult volunteers and were cultured in Teflon wells
for 5 days in RPMI 1640 (pH 7.4) with 20% fresh autologous serum as
previously described (17). M (
2 x
106/sample) were purified by adherence to
six-well plastic tissue-culture plates or chromic acid-cleaned glass
coverslips (25 mm in diameter) for 2 h at 37°C in 5%
CO2. Monolayers were washed repeatedly and then
incubated in RPMI 1640 with 2.5% autologous serum, without
antibiotics, for use in experiments. Effects of experimental
manipulations on M viability were assessed by exclusion of trypan
blue, and monolayer density was determined by nuclei counting with
napthol blue-black stain (17, 18).
Infection of M with M. tuberculosis and analysis
of intracellular survival
Monocyte-derived M in RPMI 1640, 20 mM HEPES, and 2.5% human
serum were infected with M. tuberculosis at a bacteria:M
ratio (multiplicity of infection, MOI) of 1:1 and then incubated for
1 h at 37°C. Following infection, monolayers were washed three
times with RPMI 1640 at 37°C and incubated with RPMI 1640 and 10%
FBS for 24 h, before addition of ATP or buffer control. The MOI of
1:1 was used because it permits long-term cultivation of infected human
M and the determination of intracellular viability of M.
tuberculosis at 24 h following infection. We have previously
used higher levels of MOI, including 10:1, 30:1, and 100:1, but the
resultant rapid lysis of the M monolayer precludes accurate
determination of the bacterium’s intracellular survival
(9). M cultures were incubated at 37°C for an
additional 24 h after the addition of ATP or buffer, followed by
quantitation of the growth of M. tuberculosis by
determination of CFUs or by the Bactec method, as previously described
(6). Because primary human M exhibit a wide variance in
the number of M. tuberculosis that grow intracellularly, the
results of the CFU assay are generally presented as cumulative data
(mean ± range) expressed as a percentage of the paired control
samples. The absolute number of M. tuberculosis CFUs in the
control samples has been designated in the figure legends.
Determinations of mycobacterial viability by the Bactec method were in
excellent agreement with the results of the CFU assay (6, 19).
Determination of M [Ca2+]c and
modulation of Ca2+ levels
Calcium measurements were performed as previously described
(9). Briefly, M were adhered to collagen-coated glass
coverslips and incubated in 10 µM fura 2-acetylmethyl ester (fura 2)
in HBSS for 30 min at 37°C. Levels of
[Ca2+]c in single M,
or the mean [Ca2+]c of
groups of 10–20 cells, was determined using a Photoscan II
spectrofluorometer (Photon Technology International,
Lawrenceville, NJ).
[Ca2+]c was determined
from the ratio of fluorescence emission intensities at 510 nm following
excitation at 340 and 380 nm, respectively, as previously described
(20). To analyze the contribution of
[Ca2+]c-mediated
signaling pathways to ATP-dependent mycobactericidal activity, both
extracellular and intracellular
[Ca2+]c were
experimentally modified. Extracellular Ca2+ was
depleted by washing M twice in Ca2+-free
buffer containing 5 mM EGTA, followed by resuspension in the same
buffer and incubation for 5 min at 37°C before addition of ATP.
Inhibition of changes in
[Ca2+]c were also
produced by preincubation of M with the intracellular
Ca2+ chelator,
bis-(2-amino-S-methylphenoxy)ethane-N,N,N',N'-tetraacetic
acid tetraacetoxymethyl ester (MAPTAM) (25 µM), in EGTA-containing
buffer for 20 min at 37°C. The effects of EGTA- and MAPTAM-induced
alterations in [Ca2+]c
were verified in parallel experiments by direct determination of
[Ca2+]c in control and
treated M via fluorescence of fura 2 (20). Levels of
[Ca2+]c were <20 nM in
MAPTAM-treated cells following addition of ATP or control
Ca2+-mobilizing agonists, platelet-activating
factor (100 nM) or complement-opsonized zymosan, demonstrating the
efficacy of MAPTAM-induced
[Ca2+]c buffering
(9, 21). The effects of these modulators of extracellular
and intracellular Ca2+ on M viability and
monolayer density were determined in parallel experiments, and no
significant differences from control cells were observed. EGTA and
MAPTAM did not alter the viability of M. tuberculosis,
either when incubated directly with mycobacteria in 7H9 medium for
24 h at 37°C or when added to infected M in the absence
of ATP.
Confocal microscopy
The degree of maturation of phagosomes containing live or killed
M. tuberculosis was assessed by colocalization of the
bacilli with the acidophilic dye LysoTracker Red (Molecular
Probes, Eugene, OR) and the lysosomal protein markers cathepsin D,
CD63, and lysosome-associated membrane protein (LAMP)-1, as
described previously (9, 22). LysoTracker Red was
incubated at a 1/10,000 dilution with M monolayers in RPMI 1640, 20
mM HEPES, and 2.5% autologous serum for 2 h at 37°C.
Unincorporated dye was removed by washing, followed by infection with
M. tuberculosis. After removal of nonadherent bacilli,
LysoTracker Red was added to each well at the same concentration used
for initial labeling. At 30 min postinfection, M were fixed in
3.75% paraformaldehyde for 15 min and permeabilized with ice-cold
methanol-acetone (1:1). Detection of the lysosomal protein markers
LAMP-1, cathepsin D, or CD63 was accomplished by incubating coverslips
with blocking buffer (PBS, 5% BSA, and 10% goat serum) for 1 h,
followed by the appropriate 1° Abs (diluted in blocking buffer) for
1 h, repeated washings, and incubation with Texas Red-conjugated
secondary anti-IgG Ab for 1 h, all at 25°C. The localization
of M. tuberculosis was determined by incubating monolayers
with auramine for 20 min at 25°C, followed by a 3-min incubation in
0.5% acid alcohol. Following repeated washings, coverslips were
mounted with buffered glycerol solution and nail polish.
Confocal microscopy was performed on a Zeiss Laser Scan inverted 510
microscope (Zeiss, Oberkochen, Germany). An argon-krypton laser
(excitation, 488 nm; emission band pass, 505–530 nm) was used for
detection of auramine fluorescence, and a helium-neon laser
(excitation, 543 nm; emission limit of pass, 585 nm) was used
for detection of Texas Red and LysoTracker Red. The percentage of
M. tuberculosis phagosomes colocalizing with the marker of
interest was determined by counting 25 phagosomes from each sample. The
effects of modulation of M
[Ca+2]c on the response
to ATPe was performed by preincubation with EGTA
or MAPTAM, respectively. Neither EGTA nor MAPTAM directly affected the
fluorescence of auramine, LysoTracker Red, or Texas Red
(9). At a MOI of 1:1, <10% of M contained two or more
bacteria. Therefore, confocal images of M containing single bacilli
are shown as most representative.
For quantitative analysis of phagosomal maturation, the range of
fluorescence intensity of the Texas Red 2° Ab or LysoTracker Red was
recorded along the major axis of the bacillus (defined by auramine
staining). Samples in which the mean fluorescent intensity of the given
lysosomal marker was greater than the mean of control, uninfected M
were scored as positive. For a given phagosome to be scored as a mature
phagolysosome, each of the four lysosomal markers (LAMP-1, cathepsin D,
CD63, and LysoTracker Red) must have all been positive. Because the
confocal measurements were acquired in a blinded fashion, inherent
limitations of the technique (e.g., the lack of a single, fully
specific marker of M lysosomes) should be distributed equally among
the experimental groups. The use of this technique by our laboratory
(9, 22) and others (23, 24, 25) has resulted in
strong interobserver correlations, as well as excellent agreement with
the results of immunoelectron microscopy (25, 26, 27).
Data analysis
Data from each experimental group were subjected to an analysis of normality and variance. Differences between experimental groups composed of normally distributed data were analyzed for statistical significance using Student’s t test. Nonparametric evaluation of other data sets was performed with the Mann-Whitney rank sum test. Analysis of correlation was performed with the Spearman rank order test (28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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requires increases in
[Ca2+]c
To characterize the biochemical determinants of
ATPe-stimulated killing of intracellular M.
tuberculosis, we used an in vitro model system in which blood
monocytes are differentiated to a M phenotype by cultivation for 5
days, followed by purification via adherence to tissue culture plates
(6, 17, 22). M were infected with the virulent H37Rv
strain of M. tuberculosis, at a MOI of 1:1 for 24 h,
followed by incubation with 3 mM ATP. Treatment with
ATPe reduced the viability of intracellular
M. tuberculosis by 83 ± 7% at 24 h and by
3.62 ± 0.41 logs at day 7 compared with control samples treated
with buffer alone (p < 0.001 for each time point,
n = 9) (6).
To begin to evaluate the Ca2+-dependence of this
ATPe-induced bactericidal activity, we determined
the effect of ATP on levels of
[Ca2+]c in M.
tuberculosis-infected M labeled with the fluorescent
Ca2+ indicator fura 2. ATPe
stimulated a rapid and sustained increase in
[Ca2+]c in infected M
that was indistinguishable from the
[Ca2+]c response of
uninfected cells (Fig. 1
A)
(29, 30, 31). This ATP-induced Ca2+
response was due to stimulation of two classes of M cell surface
purinergic receptors, P2Y2,
G protein-coupled receptors and
P2X7, pore-forming
receptors, because the
P2Y2-selective agonist,
UTP, produced the initial rapid increase, but not the prolonged
elevation, in [Ca2+]c
(Fig. 1B), whereas the
P2X7-selective agonist
3'-O-(4-benzoyl)benzoyl-ATP, produced a delayed but
sustained increase in
[Ca2+]c in the absence of
the initial rapid response (Fig. 1C) (30). To
determine the source of the increase in
[Ca2+]c following
stimulation by ATPe, we used the extracellular
Ca2+ chelator EGTA as well as the intracellular
Ca2+ buffer MAPTAM (9). Incubation
of M in Ca2+-free medium containing 3 mM EGTA
significantly decreased the magnitude and duration of the
ATPe-induced increase in
[Ca2+]c (Fig. 1D). Addition of the intracellular
Ca2+ chelator MAPTAM (25 µM) to M in
EGTA-containing medium completely eliminated any change in
[Ca2+]c in response to
ATPe (Fig. 1D).
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24 h after addition of ATP.
EGTA treatment reduced the level of mycobacterial killing by 83 ±
6% (Fig. 2A, p
< 0.001 and n = 7). M incubated with MAPTAM and
EGTA (which exhibit no elevation of
[Ca2+]c in response to
ATPe) demonstrated complete inhibition of
ATP-induced bactericidal activity toward H37Rv M.
tuberculosis (Fig. 2A, p < 0.001 and
n = 7). To verify the results of the CFU assay for
mycobacterial viability, we used a complimentary Bactec method based on
mycobacterial-specific oxidation of
[14C]palmitate to
[14C]CO2. These studies
confirmed the Ca2+-dependence of
ATPe-induced reductions in the intracellular
viability of H37Rv M. tuberculosis (Fig. 2B).
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B, p
< 0.001 for each strain and n = 5). Taken together,
these data demonstrate that ATPe-induced killing
of virulent M. tuberculosis within human M requires an
increase in [Ca2+]c.
Because specific stimulation of M
P2Y2 receptors via UTP does
not result in killing of intracellular M. tuberculosis
(6), these data are consistent with a requirement for
P2X7-mediated influx of
extracellular Ca2+ for tuberculocidal
activity. ATP induces Ca2+-dependent maturation of M. tuberculosis phagosomes
The specific antimicrobial mechanism(s) by which
ATPe induces intracellular mycobacterial killing
are unknown (6, 8). Because Ca2+
regulates several phagocyte antimicrobial responses including
generation of reactive oxygen species (33) and reactive
nitrogen intermediates (34, 35), granule secretion
(36), synthesis of cytokines (37), and, in
certain cases, phagosome-lysosome (P-L) fusion (9), the
Ca2+-dependence of
ATPe-induced killing of M.
tuberculosis may be due to multiple effects. We tested the
hypothesis that increases in
[Ca2+]c promote
ATPe-induced killing of M.
tuberculosis via enhancement of P-L fusion based on the following
rationale: 1) During initial infection of naive human M by M.
tuberculosis, mycobacterial inhibition of M
Ca2+ signaling is tightly coupled to inhibition
of P-L fusion and promotion of intracellular bacterial viability
(9); 2) pharmacologic reversal of M.
tuberculosis-induced inhibition of M Ca2+
signaling results in increased levels of both P-L fusion and
mycobacterial killing (9); and 3)
ATPe-stimulated killing of BCG does not involve
reactive oxygen or reactive nitrogen intermediates species and has not
been linked to synthesis or secretion of inflammatory mediators by
infected M (8). Concerning the first two points above,
we recognize that biochemical mechanisms that regulate intracellular
viability of M. tuberculosis during primary infection of
naive M may be distinct from those mechanisms involved in
ATPe-mediated reductions in mycobacterial
viability following established infection of M.
To test the hypothesis that the Ca2+ requirement
in ATPe-induced mycobacterial killing is due its
promotion of P-L fusion, we determined levels of phagosomal maturation
in control M as well as in those treated with ATP with or without
MAPTAM/EGTA. The extent of maturation of M.
tuberculosis-containing phagosomes was quantitated by laser
scanning confocal microscopy using three protein markers of
lysosomes/late endosomes (CD63, cathepsin D, and LAMP-1) and the
acidophilic dye LysoTracker Red (9). Although there is no
single, unambiguous marker for lysosomes, the relative accumulation of
several distinct proteins (both soluble and membrane associated) and
pH-sensitive dyes provides accurate determination of the identity of
vesicular compartments (22, 25, 26, 27, 29, 38). As previously
demonstrated, phagosomes containing live H37Rv M.
tuberculosis exhibited low levels of the lysosomal markers,
consistent with an immature maturational state, i.e., inhibition of P-L
fusion (Fig. 3). Incubation with ATP for
30 min resulted in marked increases in each of the lysosomal markers,
consistent with promotion of P-L fusion. Inhibition of
ATPe-induced increases in
[Ca2+]c with MAPTAM/EGTA
resulted in a complete reversal of ATP-stimulated phagosomal maturation
(Fig. 3). In fact, the level of lysosomal markers on M.
tuberculosis phagosomes in M incubated with MAPTAM/EGTA
followed by ATP was lower than that present in control M. These data
support the novel hypotheses that ATPe-induced
antituberculous activity involves the promotion of P-L fusion, and the
obligatory role of Ca2+ in
ATPe-stimulated mycobacterial killing is due, at
least in part, to the Ca2+ dependence of P-L
fusion.
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was highly correlated with P-L fusion (defined as presence of all
four lysosomal markers; r = 0.916, p
< 0.001, and n = 9). Similarly, the degree of P-L
fusion correlated highly with reductions in M. tuberculosis
viability (r = 0.926 and p < 0.001).
Finally, ATPe-induced levels of
[Ca2+]c correlated highly
with reductions in mycobacterial viability (r = 0.942
and p < 0.001). Although these high degrees of correlation
between each of the variables do not prove a causal connection, they
strongly support our hypothesis. ATP-induced killing of intracellular M. tuberculosis exhibits distinct requirements for both Ca2+ and PLD activity
We have recently demonstrated that
ATPe-stimulated killing of M.
tuberculosis within human M is tightly coupled to activation of
the PLD signal transduction pathway (6). Therefore, we
sought to determine whether the requirements for increases in
[Ca2+]c and PLD activity
were independent functions of distinct signaling pathways or whether
they exhibited some degree of interdependence. The importance of this
distinction arises from the fact that both
Ca2+-dependent PLD activities as well as
PLD-dependent increases in
[Ca2+]c have been
previously demonstrated (10, 39).
To determine whether the ATPe-induced increases
in M [Ca2+]c require
stimulation of PLD activity, we used two well-characterized,
mechanistically distinct inhibitors of PLD activity,
2,3-diphosphoglycerate (2,3-DPG) and ethanol. Although no specific
pharmacologic inhibitor of PLD has been identified, the combined use of
these complementary inhibitors has yielded substantial insights into
the role of PLD in diverse physiologic functions (6, 17).
2,3-DPG is a competitive inhibitor of PLD that exhibits low toxicity to
intact cells (17, 40). Preincubation of M monolayers
with 2,3-DPG (0.1–3 mM) for 15 min at 37°C produced dose-dependent
inhibition of ATP-stimulated PLD activity (6) with a
maximal reduction of 91% (range 87–93% reduction, p
< 0.001 and n = 5) at 5 mM 2,3-DPG. Despite this
marked inhibition of ATPe-stimulated PLD
activity, 2,3-DPG had no detectable effect on the accompanying increase
in [Ca2+]c (Fig. 4A).
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with
ethanol (0.03–1.0%, v/v) for 2 min at 37°C before addition of ATP
resulted in concentration-dependent inhibition of phosphatidic acid
generation (6) with a 93% reduction produced by addition
of 1.0% ethanol (between 89 and 95% reduction, p < 0.001
and n = 5). Similar to the results with 2,3-DPG,
ethanol had no significant effect on
ATPe-stimulated increases in M
[Ca2+]c (Fig. 4A). At the concentrations used, 2,3-DPG and ethanol had no
effect on M viability or monolayer density (data not shown). Taken
together, the results with 2,3-DPG and ethanol demonstrate that
ATPe-induced increases in M
[Ca2+]c are independent
of the accompanying stimulation of PLD activity.
To evaluate whether ATP-stimulated increases in PLD activity were
dependent on [Ca2+]c,
M were radiolabeled with [3H]oleate for
18 h at 37°C. Following removal of unincorporated label, M
were incubated in various concentrations of extracellular
Ca2+ (buffered with EGTA) before addition of 3 mM
ATP. PLD activity was determined by accumulation of the PLD-specific
product PEt in the presence of 0.5% ethanol. In M incubated in 25
µM MAPTAM/3 mM EGTA, ATPe stimulated
significant levels of PLD activity (Fig. 4B). Increases in
extracellular
[Ca2+]c resulted in
progressive increases in ATPe-stimulated PLD
activity. Thus, Ca2+ was not required for
activation of PLD but, rather, augmented the level of PLD activity
stimulated by ATPe. These data on the
relationship between Ca2+ and PLD activity in
M. tuberculosis-infected human M are in close agreement
with previous work by el-Moatassim and Dubyak in uninfected murine M
(30, 42).
Our previous report (6) and the current data strongly
suggest that ATPe-induced killing of
intracellular M. tuberculosis requires both elevation of
[Ca2+]c and activation of
PLD. Furthermore, ATPe-stimulated bactericidal
activity is tightly coupled to
[Ca2+]c-dependent promotion of P-L
fusion. Thus, we hypothesize that activation of PLD may also be
required for maturation of the M. tuberculosis-containing
phagosomes to phagolysosomes. To test this hypothesis, levels of P-L
fusion were determined in M in which PLD activity was inhibited by
2,3-DPG, ethanol, or MAPTAM/EGTA. In parallel experiments, the
viability of intracellular M. tuberculosis was determined to
assess its correlation with the extent of P-L fusion. We have
previously reported the dose-dependent inhibition of
ATPe-stimulated PLD activity by 2,3-DPG and
ethanol (6). In the current experiments, specific
concentrations of these inhibitors were selected that either maximally
inhibited ATPe-induced PLD activity (5 mM
2,3-DPG, 1% ethanol) or inhibited PLD activity to the same extent as
25 µM MAPTAM/3 mM EGTA (2 mM 2,3-DPG, 0.5% ethanol), i.e., 40%
of the maximum (Fig. 4B).
Inhibition of ATPe-stimulated PLD activity (Fig. 5A) was accompanied by
concordant reductions in P-L fusion (Fig. 5B) and inversely
proportional increases in M. tuberculosis viability (Fig. 5C). These data support the hypothesis that
ATPe-induced stimulation of PLD activity is
coupled to promotion of P-L fusion and reductions in intracellular
viability of M. tuberculosis. Of note, incomplete inhibition
of PLD activity by 2 mM 2,3-DPG or 0.5% ethanol (to the level of PLD
activity present in MAPTAM-treated M) resulted in partial inhibition
of P-L fusion (Fig. 5B) and partial restoration of
mycobacterial viability (Fig. 5C). These results contrast
with the complete inhibition of ATPe-stimulated
P-L fusion and mycobacterial killing in MAPTAM-treated M, in which
[Ca2+]c was completely
inhibited. Taken together, these data support the hypothesis that
increases in PLD activity and
[Ca2+]c make distinct and
experimentally separable contributions to ATP-induced P-L fusion and
mycobacterial killing.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are central to each of the complex stages of tuberculosis,
from initial infection through the development of active disease. A
crucial feature of pathogenesis is the ability of tubercle bacilli to
evade the microbicidal activities of M and to persist as
intracellular parasites within membrane-enclosed vesicles (4, 5). Following primary infection, the M.
tuberculosis-containing phagosome exhibits characteristics of
early, recycling endosomes and fails to progress along the physiologic
maturation pathway to a bactericidal phagolysosome (25, 26, 27, 38, 43). Little information is available concerning the
intracellular compartment in which the bacilli reside as the duration
of infection increases. Knowledge of the biochemical composition and
regulatory determinants of these intracellular havens is essential to
the development of rational, targeted interventions to modify the
survival of M. tuberculosis within M. Therapeutic
augmentation of host immunity could contribute both to the treatment of
patients with active disease, as well to the prevention of tuberculosis
in the third of the world’s population (2 billion persons) latently
infected with M. tuberculosis (1, 2).
We have recently demonstrated that ATPe
stimulates human M to kill intracellular virulent M.
tuberculosis (6). ATPe-induced
restriction of mycobacterial viability requires stimulation of M
cell surface P2X7 receptors
and is tightly coupled to stimulation of host PLD activity (6, 8). However, the M bactericidal mechanism(s) that directly
kills intracellular M. tuberculosis is unknown, as is its
relation to occupancy of
P2X7 receptors and
activation of PLD. In this paper, we present several novel features of
ATP-induced killing of virulent M. tuberculosis in human
M. First, the mycobactericidal effect of ATPe
required elevation of
[Ca2+]c based on the
significant inhibition by the extracellular Ca2+
chelator, EGTA, and complete inhibition by the intracellular
Ca2+ buffer, MAPTAM. Second, incubation of
infected M with ATP resulted in the maturation of M.
tuberculosis phagosomes to phagolysosomes, reversing the
characteristic inhibition of P-L fusion that is a hallmark of
tuberculous pathogenesis. Third, ATPe-induced
increases in [Ca2+]c and
PLD activity exert distinct and complementary roles in the induction of
P-L fusion and killing of intracellular M. tuberculosis.
Specifically, ATPe-stimulated increases in
[Ca2+]c are independent
of PLD, and Ca2+ is not required for
ATPe-induced activation of PLD (although
micromolar concentrations of Ca2+ maximally
augment the level of ATPe-stimulated PLD
activity).
The requirement for increases in
[Ca2+]c and PLD activity
for both P-L fusion and restriction of the intracellular viability of
M. tuberculosis in ATPe-stimulated
M supports the hypothesis that phagosomal maturation directly
contributes to mycobactericidal activity. These data are also
consistent with the corollary hypothesis that the requirements for
Ca2+ and PLD activity in ATP-induced
antimycobacterial activity are due to the Ca2+
and PLD dependence of P-L fusion. Further testing of these hypotheses
is currently hampered by our limited understanding of the molecular
mechanisms of tuberculous pathogenesis. For example, lack of
information regarding the biochemical signals that regulate the
maturation of M. tuberculosis phagosomes hinders the
identification of compounds that might inhibit P-L fusion in a
Ca2+- and PLD-independent manner. If available,
such inhibitory compounds could be used to directly test whether P-L
fusion is required for ATP-induced reductions in mycobacterial
viability. Similarly, because the specific mechanism(s) by which P-L
fusion leads to killing of M. tuberculosis in human M is
unknown, it is not yet possible to evaluate the
Ca2+ and PLD dependence of specific
mycobactericidal reactions.
Our previous work (9, 22) and the results presented herein
demonstrate that [Ca2+]c
regulates the intracellular viability of M. tuberculosis in
two different experimental models designed to represent distinct stages
in the host-pathogen interaction. In the first case, primary infection
of naive human M, the intracellular survival of M.
tuberculosis requires inhibition of M
Ca2+ signaling, which closely correlates with
inhibition of P-L fusion (9, 22). Transient (20 min)
pharmacologic elevation of
[Ca2+]c at the time of
infection results in P-L fusion and decreased viability of
intracellular bacilli. In the second case (this paper), in M
stably infected with M. tuberculosis for 24 h (Figs. 3 and 5) to 7 days (data not shown), elevation of
[Ca2+]c is required for
ATPe-induced P-L fusion and reduction in the
intracellular survival of tubercle bacilli.
Characterization of the Ca2+ dependence of
mycobacteria-M interactions in these two distinct experimental
models will require more detailed kinetic analysis and further
identification of the specific target proteins (22) that
directly mediate killing of intracellular M. tuberculosis
during both primary and established infection of human M. We have
recently demonstrated that Ca2+-dependent killing
of M. tuberculosis during the initial infection of M
requires specific activation of calmodulin and calmodulin-dependent
protein kinase II on the phagosomal membrane (22). Studies
to determine whether this signal transduction pathway functions in
ATP-induced killing of M. tuberculosis following stable
infection of human M are in progress. The current data,
demonstrating that mycobacterial phagosomes exhibit
Ca2+-regulated maturation as late as 7 days
following infection, has novel and important implications from the
perspectives of both pathogenesis and therapeutics. Regarding the
former, these findings establish Ca2+ as the
first known molecular regulator of phagosome physiology at this late
time point. Furthermore, the data suggest a certain degree of stability
of the phagosomal phenotype over, at least, the first week of
infection. Regarding the later point, this study furthers our
understanding of the molecular mechanisms that regulate ATP-dependent
killing and supports the general therapeutic model that physiologic
host immune defenses can be mobilized to treat tuberculosis. Definition
of the mechanisms by which physiologic compounds such as ATP
(6, 7, 8), cytokines (23), and T cell components
(44, 45) exert mycobactericidal activity may provide
important therapeutic advances, which are particularly critical due to
increasing antimicrobial resistance in M. tuberculosis.
Because M. tuberculosis is a virulent pathogen specific to
humans, it is difficult to estimate the degree to which results with
murine M or attenuated strains (e.g., BCG) correlate with human
immunity in tuberculosis. For example, the ability of
ATPe to stimulate killing of M.
tuberculosis in murine M that lack
P2X7 receptors may indicate
the presence of additional bactericidal mechanisms in normal mice.
Alternatively, it may be due to the acquisition of compensatory
mechanisms in mice that develop in the absence of the
P2X7 receptor, that normally regulates several
critical M functions, including transcription, secretion, giant cell
formation, and apoptosis (8, 14, 15, 16, 25). A second example
of a result obtained from a different experimental system that
conflicts with our data is the demonstration that
ATPe-induced killing of the attenuated BCG strain
is Ca2+ independent (8). This
difference in the requirement for Ca2+ between
our studies may be due to the operation of distinct regulatory
mechanisms involved in M that encounter a virulent pathogen
(M. tuberculosis, this study) vs an attenuated vaccine
strain (8). Additionally, differences in experimental
methods may have contributed to the conflicting results, because
intracellular Ca2+ chelators, which can
completely inhibit agonist-dependent increases in
[Ca2+]c, were not used in
the BCG study (8). Molecular definition of the
Ca2+-dependent steps in the killing of
intracellular M. tuberculosis will be required to clarify
these points and to establish the specific similarities and differences
in the interactions of human M with M. tuberculosis
vs BCG.
Note added in proof.
During revision of this manuscript, Lammas and colleagues (8) reported, in contrast to their earlier findings, that ATP-induced killing of BCG is Ca2+ dependent and is associated with phagosomal acidification (46).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David J. Kusner, Division of Infectious Diseases, Department of Internal Medicine, University of Iowa, 200 Hawkins Drive SW 54-I, GH, Iowa City, IA 52242. E-mail address: david-kusner{at}uiowa.edu
3 Abbreviations used in this paper : M, macrophage; ATPe, extracellular ATP; BCG, bacille Calmette Guérin; PLD, phospholipase D; [Ca+2]c, cytosolic Ca+2 concentration; MOI, multiplicity of infection; MAPTAM, Bis-(2-amino-S-methylphenoxy) ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester; LAMP, lysosome-associated membrane protein; P-L, phagosome-lysosome; 2,3-DPG, 2,3-diphosphoglycerate; PEt, phosphatidylethanol; fura 2, fura 2-acetylmethyl ester.
Received for publication March 22, 2001. Accepted for publication July 10, 2001.
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), and
levels of [Ca2+]c were monitored via fura 2
fluorescence. Infected M
), 3 mM ATP (
), or 25 µM MAPTAM plus 3 mM EGTA
followed 20 min later by 3 mM ATP (
). Viability of each strain was
determined by the Bactec method and expressed as the growth index
(mean ± SEM). Data in A and B
represent results from five identical experiments, each performed in
triplicate.
