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,
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Inflammation Program,
Graduate Program in Immunology, and
Department of Internal Medicine, University of Iowa and Veterans Administration 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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The mechanism by which alterations in MP Ca2+ signaling are coupled to mycobacterial virulence are unknown. Based on their role in Ca2+-mediated membrane trafficking, we hypothesized that calmodulin (CaM) and Ca2+-bound CaM (Ca2+-CaM)-dependent protein kinase II (CaMKII) would be involved. CaM is a highly acidic, 17-kDa protein that functions as the predominant [Ca2+]c sensor in eukaryotic cells (11, 12). Increases in [Ca2+]c result in its binding to CaM, triggering a dramatic conformational change that exposes two critical hydrophobic patches on the surface of CaM. These hydrophobic regions of Ca2+-CaM bind to complementary hydrophobic domains present in numerous effector proteins (11, 12). Ca2+-CaM is required for fusion of endocytic vesicles with the yeast vacuole (a homologue of mammalian lysosomes), endosome-endosome fusion, receptor recycling, exocytosis, and transcytosis (13, 14, 15, 16, 17, 18, 19, 20). Because phagosomes mature via multiple fusion and fission events with vesicles of the endosomal pathway (21), CaM may regulate P-L fusion in an analogous manner. An important effector of Ca2+-CaM is CaMKII, a multifunctional Ser/Thr kinase that has recently been shown to regulate endosome-endosome fusion (14, 22, 23, 24, 25). In resting cells, CaMKII exists as an oligomeric complex of 8–12 subunits with autoinhibition of catalytic activity (26, 27). Binding of Ca2+-CaM relieves autoinhibition, resulting in intersubunit phosphorylation and activation of CaMKII (26, 27).
The objective of this study was to investigate two hypotheses: 1) infection of human MPs by M. tuberculosis is accompanied by decreased activation of CaM and CaMKII on the mycobacterial phagosome, and 2) these alterations in CaM-dependent signal transduction contribute to the inhibition of P-L fusion and intracellular survival of M. tuberculosis.
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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 (10). W5, W7, and KN-62 were obtained from Calbiochem (La Jolla, CA), and KN-04 was obtained from Bioscience Laboratories (Bethlehem, PA). The anti-CaM mAb was obtained from Sigma (St. Louis, MO), and Abs to CaMKII, including phospho-specific anti-CaMKII mAb, were obtained from Affinity Bioreagents (Golden, CO).
Preparation of MP monolayers and bacteria
PBMC were isolated from healthy, purified protein
derivative-negative adult volunteers and cultured in Teflon wells for 5
days, and MPs were purified by adherence to collagen-coated glass
coverslips as previously described (10, 28). Effects of
experimental manipulations on MP viability were assessed by exclusion
of trypan blue, and monolayer density was determined by nucleus
counting with naphthol blue-black stain. The H37Rv strain of M.
tuberculosis was cultured and prepared for use in experiments as
noted previously (10, 28). Killed (
-irradiated)
M. tuberculosis were generously provided by Drs. Patrick
Brennan and John Belisle (Colorado State University). M.
tuberculosis suspensions were counted in a Petroff-Hauser chamber,
and the concentration of bacteria was adjusted for use in experiments.
Final M. tuberculosis preparations contained >95% single
bacteria, with 
75% viability by determination of CFUs. The effects
of various experimental manipulations on the viability of M.
tuberculosis were also determined by analysis of CFUs. The
pharmacologic agents A23187,
bis-(2-amino-S-methylphenoxy)ethane-N,N,N',N'-tetraacetic
acid tetraacetoxymethyl ester (MAPTAM), W7, W5, KN-62, and KN-04 (see
below) did not alter MP viability (trypan blue exclusion), the density
of the MP monolayer (nucleus counting with naphthol blue-black)
(10), or the viability of M. tuberculosis.
Determination of MP [Ca2+]c
Calcium measurements were performed as previously described (10). Briefly, MPs were adhered to collagen-coated glass coverslips and incubated in 10 µM fura 2-AM in HBSS for 30 min at 37°C. Levels of [Ca2+]c in single MPs, or the mean [Ca2+]c of groups of 10–20 cells, was determined using a Photoscan II spectrofluorometer (Photon Technologies International, New Brunswick, NJ). The concentration of [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 (29). Pharmacologic increases in [Ca2+]c were obtained by the addition of the Ca2+ ionophore A23187 (1 µM) to MPs incubated in EGTA/CaCl2-buffered solutions. To mimic the kinetics of physiologic increases in [Ca2+]c, ionophore-induced Ca2+ elevations were reversed by the addition of phosphatidylcholine (PC) vesicles (20µg/ml) in 1% autologous serum (30). Inhibition of changes in [Ca2+]c were produced by preincubation of MPs with the intracellular Ca2+ chelator MAPTAM (25 µM) for 30 min at 37°C. The effects of A23187- and MAPTAM-induced alterations in [Ca2+]c were verified in parallel experiments by direct determination of [Ca2+]c in control and treated MPs via fluorescence of Fura2.
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 and the lysosomal protein markers cathepsin D, CD63, and lysosomal-associated membrane protein (LAMP)-1 (8, 10, 31). Lysotracker Red at a 1:10,000 dilution was incubated with MP monolayers in RPMI 1640 plus 20 mM HEPES, pH 7.4, 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, MPs 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, 10% goat serum) for 1 h, followed by the appropriate primary 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 (Oberkochen, Germany) Laser Scan Inverted 510 microscope. 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 LP, > 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 localization of CaM and CaMKII to M. tuberculosis-containing phagosomes was determined as for the lysosomal protein markers. The levels of CaM on M. tuberculosis phagosomes were quantitated by determination of the maximum fluorescence intensity (F.I.) of CaM staining along the length of the phagosome and subtracting the maximum background fluorescence value within 1 µm of the phagosome. In the case of CaMKII, both phospho-specific Ab and polyclonal Ab to a distinct epitope shared by both the phosphorylated and unphosphorylated species of CaMKII were used. Previous work has demonstrated that phosphorylated CaMKII, detected by the phospho-specific Ab, is the catalytically active form of the kinase (32, 33). The effects of elevation or chelation of MP [Ca2+]c on the phagosomal localization of CaM or CaMKII were elicited by incubation with A23187 or MAPTAM, respectively, as previously described (10). Neither A23187 nor MAPTAM directly affected the fluorescence of auramine, Lysotracker Red, or Texas Red (not shown). In experiments in which the CaM inhibitor W7 or its less potent structural analog W5 were studied, MP monolayers were treated concurrently with these compounds during the 30-min infection period.
Analysis of CFUs
MPs adherent to collagen-coated glass coverslips were infected at a multiplicity of infection (MOI) of 1:1 with preopsonized M. tuberculosis in HBSS. After 30 min, the monolayers were washed and repleted with buffer containing 1% serum. Monolayers were lysed with ice-cold sterile water, and SDS was added to a final concentration of 0.25%. Lysates obtained from the 24-h time point were combined with their corresponding supernatants and resuspended in 7H9, and serial dilutions were plated in duplicate on 7H11 agar. Colonies were counted 2 wk after plating. To determine the effect of elevation of MP intracellular Ca2+ concentration on mycobacterial survival, monolayers were infected at a 1:1 ratio with complement-opsonized M. tuberculosis in HBSS containing the Ca2+ ionophore A23187 (1 µM) or an equivalent volume of DMSO solvent (0.1%). After 20 min, monolayers were washed and repleted with 20 µg/ml PC vesicles, 1% autologous serum in RPMI 1640 plus 20 mM HEPES, pH 7.4, to reverse the A23187-mediated influx of extracellular Ca2+. The dipalmitoylphosphatidylcholine vesicles were prepared by evaporation of a chloroform:methanol (2:1) solution under N2 and resuspension in HBSS by sonication for 10 min at 25°C. To examine the effect of CaM inhibitors on the intracellular survival of M. tuberculosis in ionophore-treated cells, 1 µM KN-62 or KN-04 was added to the monolayers at the time of infection. CFUs were enumerated as described above.
Analysis of data
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 Wilcoxon Rank Sum test (34).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Elevations in
[Ca2+]c govern the
conformation, localization, and target recognition of CaM in numerous
biological systems (11, 12). Because live but not killed
M. tuberculosis infects human MPs without inducing a rise in
their [Ca2+]c
(10), we hypothesized that phagosomes containing live
M. tuberculosis would exhibit lower levels of
membrane-associated CaM compared with phagosomes surrounding dead
tubercle bacilli. To test this hypothesis, we quantified phagosomal
levels of CaM by laser-scanning confocal microscopy. PBMC isolated from
healthy, purified protein derivative-negative donors were cultured in
Teflon wells for 5 days, and monocyte-derived MPs were recovered by
adherence to collagen-coated glass coverslips. MP monolayers were
incubated with complement-opsonized live or killed (-irradiated)
M. tuberculosis at a MOI of 1:1. After 30 min, monolayers
were washed to remove nonadherent bacilli and fixed with
paraformaldehyde. Following permeabilization, infected MPs were
incubated with anti-CaM mAb followed by Texas Red-conjugated goat
anti-mouse secondary Ab. Intracellular M. tuberculosis
were localized by staining with auramine (10). Levels of
CaM on M. tuberculosis-containing phagosomes were
quantitated by determination of the maximum F.I. of the CaM signal
along the length of the phagosome, and all values were corrected for
background fluorescence (see Materials and Methods).
Phagosomes containing live M. tuberculosis (Fig. 1
, A and B)
exhibited significantly lower levels of membrane-associated CaM
compared with phagosomes surrounding killed tubercle bacilli (Fig. 1, C and D). The mean (±SEM) values for the maximal
F.I. of CaM were 87.5 ± 6.7 for live M. tuberculosis
vs 172.5 ± 16.6 for killed bacilli (p <
0.001; n = 7). For each experiment (including those
described in the remainder of this report), all cell-associated
M. tuberculosis were determined to be intracellular by
confocal microscopy. Because data from other experimental systems
indicate that the translocation of cytosolic CaM to vesicular
compartments is dependent on increases in
[Ca2+]c, we reasoned that
this difference in phagosomal CaM was due to the differential effects
of live and killed M. tuberculosis on
[Ca2+]c. To test this
hypothesis, we determined the effect of pharmacologic elevation of
[Ca2+]c on levels of
phagosomal CaM in MPs infected with live tubercle bacilli. To mimic the
transient elevation in
[Ca2+]c induced by
physiologic agonists, MPs were treated with the
Ca2+ ionophore A23187 (in buffer containing 500
µM free Ca2+) at the time of infection with
live M. tuberculosis, followed 20 min later by reversal of
ionophore function with PC vesicles. We have recently demonstrated that
this pharmacologic elevation of
[Ca2+]c results in
maturation of phagosomes containing live M. tuberculosis
into microbicidal phagolysosomes (10). The
Ca2+ ionophore caused a marked increase in
phagosomal CaM (F.I. = 186.6 ± 5.3) compared with infected MPs
treated with DMSO vehicle alone (F.I. = 87.5 ± 6.7;
p < 0.001; n = 7) (Fig. 1, A and B). These data support the hypothesis that
the decreased recruitment of CaM to membranes of phagosomes containing
live M. tuberculosis is due to the lack of elevation in
[Ca2+]c.
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, C and D). It is notable that the level of
phagosomal CaM in MAPTAM-treated MPs incubated with killed M.
tuberculosis closely approximated the level of CaM on phagosomes
from MPs infected with live mycobacteria (F.I. = 99.5 ± 17
compared with 87.5 ± 6.7; p > 0.4;
n = 7). A23187 and MAPTAM had no effect on the
viability or density of the MP monolayer or on the viability of
isolated M. tuberculosis (not shown). Taken together, these
experiments support the hypothesis that the decreased level of CaM on
phagosomes containing live M. tuberculosis is a consequence
of the lack of increase in
[Ca2+]c that accompanies
their ingestion. Inhibition of CaM blocks Ca2+-dependent maturation of M. tuberculosis-containing phagosomes
Because elevation of
[Ca2+]c is required for
both phagosomal accumulation of CaM (Fig. 1) and P-L fusion
(10), we tested the hypothesis that recruitment of CaM to
the membrane of M. tuberculosis-containing phagosomes is
necessary for P-L fusion. The naphthalene sulfonamide W7 is a
well-characterized, highly specific inhibitor of CaM that binds to the
surface-exposed hydrophobic regions of Ca2+-CaM,
preventing their interaction with complementary domains present in
target proteins (35). Incubation of A23187-treated MPs
with W7 (25 µM) significantly reduced the
Ca2+-dependent maturation of phagosomes
containing live M. tuberculosis, as determined by decreased
levels of the lysosomal protein markers cathepsin D, CD63, and LAMP-1
(Fig. 2, A and B).
Although low levels of these proteins can be detected on endosomes, the
marked increase in their relative concentrations on lysosomes has been
widely used to establish the identity of the latter compartment
(8, 9, 10, 21, 31). Furthermore, colocalization of the
acidophilic dye Lysotracker Red with M. tuberculosis was
also significantly reduced in W7-treated MPs, consistent with a
decrease in trafficking of the bacterium to phagolysosomes (Fig. 2B). These data implicate an important role for CaM in the
Ca2+-dependent reversal of the normally immature
phenotype of M. tuberculosis-containing phagosomes.
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-irradiated
M. tuberculosis acquire high levels of LAMP-1, cathepsin D,
and CD63, as well as the acidotropic dye Lysotracker Red (Fig. 2, C and D) (10). However,
preincubation of MPs with W7 inhibited the acquisition of each of these
phagolysosomal characteristics by 
70–75% (n = 4),
consistent with their dependence on Ca2+-CaM
(Fig. 2, C and D). W7 did not alter the increase
in MP [Ca2+]c stimulated
by killed M. tuberculosis (not shown). To evaluate the
specificity of pharmacologic inhibition, we conducted parallel
experiments with W5, a structural analog of W7 that is much less potent
at inhibiting Ca2+-CaM (36). In
contrast to W7, incubation of MPs with W5 (25 µM) did not inhibit the
maturation of phagosomes containing killed M. tuberculosis
(Fig. 2C). In control experiments, neither W7 nor W5 altered
the viability of MPs or isolated tubercle bacilli (not shown).
These data are consistent with a model in which: 1) elevation of
[Ca2+]c is required for
recruitment of cytosolic CaM to phagosomal membranes, and 2)
Ca2+-CaM promotes the maturation of phagosomes to
acidified phagolysosomes. Elevations in MP [Ca2+]c result in activation of CaMKII on phagosomal membranes
The Ca2+-dependent activation of CaM results in stimulation of a large number of downstream target molecules that produce diverse functional effects in various cells and tissues. We have focused on one prominent target, CaMKII, based on its established roles in the regulation of vesicular trafficking (14, 37). Our hypotheses are that: 1) activated CaMKII regulates the Ca2+-dependent maturation of M. tuberculosis phagosomes (containing either killed bacilli or live organisms in ionophore-treated MPs), and 2) that the lack of CaMKII activation following phagocytosis of live M. tuberculosis contributes to the lack of P-L fusion.
Increases in [Ca2+]c trigger the activation of CaMKII via the binding of Ca2+-CaM to a site near the autoinhibitory domain of CaMKII, which induces autophosphorylation of CaMKII at Thr286 (38). Because autophosphorylation activates CaMKII, Abs specific for phospho-Thr286 provide a sensitive means to monitor CAMKII activity in vitro and in vivo. As a first step in evaluating the potential role of CaMKII in regulating the Ca2+-dependent maturation of M. tuberculosis-containing phagosomes, we used confocal microscopy to compare the levels of total and phosphorylated (activated) CaMKII on phagosomes containing live or killed tubercle bacilli. An anti-CaMKII polyclonal Ab raised to a peptide epitope shared between the phosphorylated and unphosphorylated forms of the kinase was used to determine the total level of phagosomal CaMKII. Activated CaMKII was determined with a mAb specific for phospho-Thr286.
The level of total phagosome-associated CaMKII (phosphorylated and
unphosphorylated forms) did not differ between MPs that ingested live
or killed mycobacteria. In fact, CaMKII was detected on the membranes
of all phagosomes, regardless of the viability of the ingested M.
tuberculosis and independent of levels of
[Ca2+]c. In contrast to
levels of total CaMKII, activated CaMKII (containing
phospho-Thr286) was found much less frequently on
phagosomes containing live M. tuberculosis (mean 20%;
range, 5.0–35%; Fig. 3, A
and B) than on those encompassing killed tubercle bacilli
(mean 83.8%; range, 70–95%; n = 4; p
< 0.01; Fig. 3, C and D).
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, A and B). A23187 did not alter the phagosomal
level of total CaMKII (phosphorylated and unphosphorylated species, not
shown). To test the hypothesis that elevation of cytosolic
Ca2+ activated phagosomal CaMKII via stimulation
of CaM, we determined the effect of the CaM inhibitor W7 on levels of
autophosphorylated CaMKII. W7 (25 µM) reduced the levels of
autophosphorylated CaMKII by 65% in ionophore-stimulated cells
(p < 0.002; n = 4; Fig. 3, A and B). In contrast, W7 did not alter the
levels of total CaMKII on these phagosomes (not shown). Taken together,
these results suggest that elevations in
[Ca2+]c stimualte CaMKII
via CaM.
Conversely, in MPs ingesting killed M. tuberculosis, MAPTAM
significantly reduced the percentage of phagosomes containing activated
CaMKII (35.2%; range, 18.2–52.4%) compared with control MPs
pretreated with DMSO vehicle alone (83.8%; range, 70–95%;
p < 0.005; n = 4; Fig. 3, C
and D). Incubation of MPs with MAPTAM did not alter the
levels of total CaMKII on M. tuberculosis phagosomes (not
shown). The autophosphorylation of CaMKII accompanying ingestion of
killed M. tuberculosis could also be inhibited by the CaM
antagonist W7 (reduction from 84% (range 72–93%) to 11% (range
7–17%); p < 0.01; n = 4). In
contrast, the inactive analog, W5, had no effect on activation of
CaMKII (Fig. 3, C and D). These data support the
hypotheses that the level of
[Ca2+]c accompanying
ingestion of M. tuberculosis is an important determinant of
CaMKII activation on mycobacterial phagosomes, and that this effect of
[Ca2+]c is mediated by
Ca2+-CaM.
Activation of CaMKII is associated with maturation of M. tuberculosis-containing phagosomes and restriction of mycobacterial viability within human MPs
To investigate the hypothesis that activation of CaMKII on mycobacterial phagosomes is required for their maturation to phagolysosomes, we used the CaMKII-specific inhibitor KN-62 and its inactive structural analog KN-04 (39, 40). KN-62 is an isoquinolone sulfonamide that interferes with the binding of Ca2+-CaM to CaMKII, thus preventing the autophosphorylation-dependent activation of CaMKII (40). To first verify that KN-62 inhibited CaMKII activity in our system, we determined its effects on the autophosphorylation of CaMKII on phagosomes containing killed M. tuberculosis. As expected, KN-62 but not KN-04 significantly reduced the level of phosphorylated CaMKII on mycobacterial phagosomes (not shown).
The addition of KN-62 to A23187-treated MPs significantly reduced the
Ca2+ ionophore-dependent maturation of phagosomes
containing live tubercle bacilli, determined as marked reductions in
LAMP-1, cathepsin D, CD63, and Lysotracker Red, compared with MPs
treated with A23187 alone (Fig. 4, A and B). KN-62-mediated inhibition of CaMKII
activity also blocked the maturation of phagosomes containing killed
M. tuberculosis (Fig. 4, C and D). The
inactive structural analog KN-04 had no effect on phagosomal
maturation, regardless of the viability of the ingested M.
tuberculosis (Fig. 4, A–D). Additional control
experiments demonstrated that KN-62 and KN-04 did not affect the
viability of MPs or isolated tubercle bacilli (not shown). These
results are consistent with the following model: 1) P-L fusion requires
Ca2+-CaM-dependent activation of phagosomal
CaMKII, and 2) decreased activation of CaMKII contributes to the
inhibition of phagosomal maturation during MP infection by M.
tuberculosis.
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70% when assayed at either 30 min
or 24 h postinfection. KN-62 significantly inhibited this
Ca2+-dependent killing of intracellular M.
tuberculosis. Thirty minutes following infection, MPs treated with
both A23187 and KN-62 yielded 96 ± 3% of the CFUs derived from
control untreated MPs (Fig. 5) compared
with 34 ± 7% of the control level of CFUs following incubation
with A23187 alone (p < 0.01; n
= 4). When assayed 24 h after infection, the effect of KN-62 on
M. tuberculosis viability was still present, though to a
lesser degree. The level of mycobacterial CFUs in MPs treated with
A23187 alone was 32 ± 7% of control, whereas MPs incubated with
both A23187 and KN-62 yielded 53 ± 10% of control CFUs
(p < 0.05; n = 4). KN-04 had
no effect on the intracellular viability of M. tuberculosis
at either time point (Fig. 5). Neither KN-62 nor KN-04 altered the
extent of mycobacterial phagocytosis or the level of MP
[Ca2+]c (not shown).
Taken together, these data indicate that the level of activation of
CaMKII is an important determinant of both phagosomal maturation and
intracellular viability of M. tuberculosis within human MPs
(Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to providing novel insights into the molecular mechanisms responsible for the intracellular parasitism of M. tuberculosis within human MPs, these results represent the first demonstration of a role for CaM and CaMKII in regulating phagosome-lysosome fusion following ingestion of any particle or microbe. The molecular mechanisms by which CaM and CaMKII regulate phagosomal maturation are unknown. Review of their established roles as regulators of membrane trafficking in other biological systems suggests hypotheses regarding their functions in the analogous process of phagosomal maturation. In the yeast Saccharomyces cerevisiae, perivacuolar increases in [Ca2+]c stimulate the membrane recruitment of CaM, which is required for homotypic vacuole fusion (13). Ca2+-CaM associates with a multimolecular phosphatase complex that initiates a late step in the fusion reaction, distal to the assembly of the soluble NSF attachment receptor complex. CaM also activates phosphatidylinositol 3-kinase (43), whose product, phosphatidylinositol 3,4,5-trisphosphate, directs the association of the Rab5 effector, EEA-1, with endosomes (12, 44). EEA-1 in turn binds CaM, indicating that complex regulatory circuits involving these multiple signaling components regulate vacuole fusion (12, 44). Stahl and coworkers (14) have recently demonstrated that both CaM and CaMKII function in homotypic endosome fusion in murine MPs, an experimental system that shares many structural and functional parallels with P-L fusion. The downstream effectors of the Ca2+-CaM-CaMKII signal transduction pathway in MPs remain undefined, but may share homology with the signaling cascade of synaptic transmission, in which CaMKII-mediated phosphorylation of synaptic vesicle proteins (e.g., syntaxin-1, vesicle-associated membrane protein, synaptotagmin) regulates the docking and fusion of neurotransmitter vesicles with the presynaptic membrane (20, 37, 45).
Our focus on CaM does not preclude the possibility of additional
Ca2+-dependent effector molecules being involved
in the regulation of phagosome trafficking and bactericidal activity.
In fact, the incomplete inhibition of P-L fusion by the CaM antagonist
W7 is consistent with a requirement for additional regulatory elements
(Fig. 2). Similarly, inhibition of CaMKII by KN-62 resulted in nearly
complete reversal of Ca2+ ionophore-induced
restriction of mycobacterial growth at 30 min, but only partially
restored intracellular viability at the 24-h time point (Fig. 5). This
dichotomy suggests that CaMKII-independent effects are involved in
ionophore-induced reductions in the intracellular survival of M.
tuberculosis, particularly at later time points. Another class of
potential Ca2+-regulated modulators of phagosomal
maturation are the annexins, a family of
Ca2+-dependent phospholipid-binding proteins that
are highly enriched on endosomes and regulate homotypic endosome fusion
(46, 47). In human neutrophils, phagocytosis of the
attenuated H37Ra strain of M. tuberculosis also occurs in
the absence of a change in
[Ca2+]c, and the
resultant phagosome exhibits a distinctive profile of annexin isoforms,
compared with phagosomes encompassing particles that stimulate an
increase in [Ca2+]c
(48). Further definition of the role of annexins in the
maturation of mycobacterial phagosomes in neutrophils and MPs will
likely provide additional insight into the mechanisms by which
elevations in [Ca2+]c
regulate P-L fusion and bactericidal activity. Phagocyte production of
reactive oxygen species (ROS) by the NADPH oxidase is also regulated by
changes in [Ca2+]c.
Because ROS function both as bactericidal compounds and as signal
transduction intermediates that regulate multiple effector pathways of
phagocytes and other cells (49), the role of ROS in
mycobacterial pathogenesis and its relation to alterations in
Ca2+ signaling will require careful analysis.
The requirement for elevations in [Ca2+]c for P-L fusion in human MPs (this paper, and Ref. 10) differs from that previously reported by Zimmerli et al. (50). If the Ca2+ requirement for MP P-L fusion demonstrates some degree of particle specificity, this could account for the differences between our studies. We used live and dead virulent H37Rv M. tuberculosis because of the well-established differences in intracellular trafficking of the resultant phagosomes, with their important implications for mycobacterial pathogenesis (6, 8, 10). Zimmerli et al. (50) used Mycobacterium bovis bacillus Calmette-Guérin, coagulase-negative staphylococci, and complement-opsonized zymosan. A microbe-specific property that may modulate the biochemical requirements for P-L fusion is the organism’s virulence. The H37Rv strain of M. tuberculosis is a virulent intracellular pathogen, whereas bacillus Calmette-Guérin is a markedly attenuated vaccine strain; coagulase-negative staphylococci are extracellular pathogens of low virulence; and zymosan is a polysaccharide-rich preparation from the cell wall of S. cerevisiae. Other potential reasons for the difference in results between these studies are the respective methods of measuring phagosomal maturation. Zimmerli et al. (50) defined maturation of phagolysosomes by colocalization of the ingested particles with LAMP-1 and endocytosed rhodamine dextran. In our previous study (10) and in this report, we used three protein markers (CD63, cathepsin D, and LAMP-1) and the acidophilic dye Lysotracker Red to characterize the maturation of mycobacterial phagosomes. Because late and even early endosomes share certain protein constituents with lysosomes (e.g., staining for LAMP-1 and accumulation of extracellular dextran), determination of the identity of vesicular compartments depends on the relative concentrations of these markers rather than their mere presence or absence (6, 8, 9, 10, 21, 31). In light of these complexities, it is likely that additional approaches, such as in vitro reconstitution, will be required to clarify the Ca2+ requirement for P-L fusion in MPs.
Our analysis of the functional consequences of defective activation of CaM and CaMKII (inhibition of P-L fusion and promotion of intracellular survival) relied on the use of well-characterized, specific small-molecule inhibitors (W7 and KN-62) (12, 25, 35, 36, 39, 40). This experimental approach was primarily due to the technical limitations of using primary human monocyte-derived MPs, which are recalcitrant to genetic manipulation, for our in vitro model of M. tuberculosis infection. Control experiments with inactive structural analogs (W5 and KN-04) supported the specificity of inhibition. Because phosphorylation of Thr286 correlates directly with CaMKII activity (25, 26, 27, 51), the demonstration that KN-62-mediated reductions in phospho-Thr286 correlated with inhibition of P-L fusion and mycobacterial killing provided an internal control of the efficacy of this inhibitor. Similarly, inhibition of the activity of an established Ca2+-CaM-dependent enzyme (in this case, CaMKII) has been widely used as a mechanistically defined correlate of pharmacologic inhibition of CaM (12, 14, 35). Taken together, these considerations demonstrate that at the concentrations used, W7 and KN-62 inhibited CaM and CaMKII, respectively, and strongly suggest that nonspecific effects were minimal.
The potential relevance of CaM and CaMKII to the antimicrobial activities of MPs toward pathogens other than M. tuberculosis is currently under evaluation. It is worth noting that, like M. tuberculosis, numerous intracellular bacterial and protozoal pathogens use complement receptors to invade MPs, and in several cases failure of phagosomal maturation has been documented (10, 52, 53). CaM may represent an attractive target for pathogens because it regulates a number of signaling components involved in endosome trafficking and membrane fusion. In addition to the present focus on the roles of CaM and CaMKII in phagosome maturation, Ca2+ is a ubiquitous, multifunctional second messenger that regulates diverse phagocyte antimicrobial responses (54, 55, 56). In fact, infection of MPs by M. tuberculosis is accompanied by numerous defects in the orchestration of the innate and acquired immune responses, including decreased Ag presentation, aberrant cytokine secretion, and decreased detection of ROS (3, 4, 41). As with the majority of its effects on the immune system, the components of M. tuberculosis responsible for alterations in CaM-dependent signal transduction are unknown. Further investigation of the roles of CaM and CaMKII in MP immune responses should contribute both to our specific understanding of the intracellular pathogenesis of M. tuberculosis and to our fundamental knowledge of phagocyte antimicrobial defenses.
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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 34-I, GH, Iowa City, IA 52242.
3 Abbreviations used in this paper: MP, macrophage; [Ca2+]c, cytosolic Ca2+; CaM, calmodulin; Ca2+-CaM, Ca2+-bound CaM; CaMKII, Ca2+-CaM-dependent protein kinase II; MAPTAM, bis-(2-amino-S-methylphenoxy) ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester; P-L, phagosome-lysosome; PC, phosphatidylcholine; F.I., fluorescence intensity; MOI, multiplicity of infection; ROS, reactive oxygen species; LAMP, lysosomal-associated membrane protein.
Received for publication September 28, 2000. Accepted for publication December 18, 2000.
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or mAb to
phospho-Thr286 (
). Texas Red-conjugated secondary Abs
were used for detection of the anti-CaMKII Abs, and M.
tuberculosis was stained with auramine. A and
C are representative confocal images, whereas
B and D present cumulative data
(mean ± range) from 25 phagosomes per sample, from four different
experiments.
