From the Division of Cell Biology, Hospital for Sick
Children, Toronto, Ontario M5G 1X8, Canada, the
Department
of Medical Genetics and Microbiology, University of Toronto, Toronto,
Ontario M5S 1A8, Canada, and ** Takeda Chemical
Industries Ltd., Osaka, 540-8645 Japan
Received for publication, November 21, 2002, and in revised form, January 31, 2003
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ABSTRACT |
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Neisseria gonorrhoeae can be
internalized by mammalian cells through interactions between bacterial
opacity-associated (Opa) adhesins and members of the human
carcinoembryonic antigen-related cellular adhesion molecule (CEACAM)
family. We examined the role of phosphatidylinositol 3-kinases
(PI3Ks) in gonococcal invasion of epithelial cell lines
expressing either CEACAM1 or CEACAM3. CEACAM3-mediated internalization,
but not that mediated by CEACAM1, was accompanied by localized and
transient accumulation of the class I PI3K product phosphatidylinositol
3,4,5-trisphosphate at sites of bacterial engulfment. Inhibition
of phosphatidylinositol 3-kinases reduced CEACAM3-mediated uptake but,
paradoxically, led to an increase in intracellular survival of bacteria
internalized via either CEACAM1 or CEACAM3, suggesting additional roles
for PI3K products. Consistent with this finding, the class III PI3K product phosphatidylinositol 3-phosphate accumulated and persisted in
the membrane of gonococcal phagosomes after internalization. Inhibition
of PI3K blocked phagosomal acquisition of the late endosomal marker
lysosome-associated membrane protein 2 and reduced phagosomal
acidification. Inhibiting phagosomal acidification with
concanamycin A also increased survival of intracellular gonococci. These results suggest two modes of action of
phosphatidylinositol 3-kinases during internalization of
gonococci: synthesis of phosphatidylinositol 3,4,5-trisphosphate is
important for CEACAM3-mediated uptake, while phosphatidylinositol
3-phosphate is needed for phagosomal maturation and acidification,
which are required for optimal bacterial killing.
Gonorrhea is typified by a urethral or cervical exudate that
predominantly consists of polymorphonuclear neutrophils with intracellular and extracellular associated Neisseria
gonorrhoeae. Gonococci interact with various human cell types,
including mucosal epithelia, endothelia, and immune cells, through a
number of adhesins. Most colony opacity-associated
(Opa)1 protein variants
mediate tight bacterial attachment by binding to various members of the
carcinoembryonic antigen-related cellular adhesion molecule (CEACAM)
family. Four of these, CEACAMs 1, 3, 5, and 6, can act as receptors for
bacteria expressing these adhesins. CEACAM1 is the most broadly
distributed, being expressed in a variety of tissues and cell types
where it can support bacterial invasion into and trafficking across
epithelial layers (1), entry into primary endothelial cells (2), and
engulfment by professional phagocytes (3). In contrast, CEACAM3 is
expressed exclusively by neutrophils and supports the efficient
non-opsonic uptake of Opa-expressing bacteria by a process reminiscent
of that mediated by phagocytic Fc receptors (4). This
CEACAM3-dependent uptake may constitute an important
component of host innate immune defense against gonorrhea.
While binding of Opa-expressing bacteria to CEACAM receptors can lead
to internalization of bacteria into the host cell, the molecular
mechanisms underlying this uptake are poorly understood. The fate of
gonococci after internalization and host cell factors that influence
that fate are also unclear.
Phosphatidylinositol 3-kinases (PI3Ks) play a key role in signaling
downstream of numerous receptors and in orchestrating a range of
membrane traffic events (5-7). These kinases generate phosphoinositides bearing phosphate on the 3-position of the inositol ring. The resulting lipid second messengers can be recognized by a
variety of effector proteins, leading to changes in membrane and
cytoskeletal dynamics (8). Of note, PI 3-kinases have been implicated
in bacterial invasion of epithelial cells by Listeria monocytogenes (9) and Helicobacter pylori (10). In
contrast, invasion of epithelial cells by Salmonella
typhimurium is PI3K-independent (11). PI3K activity is also
required for efficient phagocytosis of antibody-coated particles by
macrophages, a process of engulfment that is similar in several
respects to some forms of bacterial invasion (12, 13).
Given the importance of PI 3-kinases in numerous instances of membrane
and cytoskeletal remodeling and our limited understanding of the
mechanisms of Opa-dependent gonococcal uptake into human cells, we investigated whether PI 3-kinases are involved in uptake of
gonococci via CEACAM receptors. To this end, we used human epithelial
cells stably transfected with individual CEACAM receptors, a model
system that has been used successfully before (3, 4). By expressing
single CEACAM receptors in isolation, the reconstituted system
circumvents complications arising from the co-expression of multiple
CEACAM receptors in native cells. It also has the advantage that the
cells are amenable to transfection, whereas primary human cells such as
neutrophils are refractory to such manipulations.
Our results reveal major differences in phosphoinositide dynamics
during gonococcal internalization depending on the CEACAM receptor
used. In addition, distinct 3-phosphoinositides are involved at
different stages during gonococcal internalization and the subsequent
maturation of the gonococcal phagosome.
Reagents and Plasmids--
Wortmannin and LY294002 were from
Calbiochem. TRITC and FITC were from Molecular Probes. Gentamicin was
from Bioshop Canada Inc. (Burlington, Ontario, Canada). GC agar
and Isovitalex were from BD Biosciences. Concanamycin A was from Sigma.
Anti-gonococcal antibody UTR01 was prepared as described
previously.2 Anti-LAMP-2
antibody H4B4 was from the Developmental Studies Hybridoma Bank
(University of Iowa). Cy3- and Cy5-conjugated secondary antibodies were
from Jackson ImmunoResearch Laboratories. Alexa488- and Texas
Red-conjugated secondary antibodies and Alexa633-phalloidin were from
Molecular Probes. Plasmids encoding chimeras of green fluorescent
protein (GFP) with the PH domain of Akt, with two tandem FYVE domains,
and with the PX domain of p40phox have been described
previously (14-16).
Cell Culture and Bacterial Growth--
HeLa cells stably
expressing CEACAM1 and CEACAM3 have been described previously (3).
HeLa-CEACAM1 and HeLa-CEACAM3 cells were grown in RPMI 1640 medium with
10% fetal calf serum. Transient transfections of these cells were
performed with FuGENE 6 (Roche Molecular Biochemicals) according to the
manufacturer's instructions with overnight incubations for plasmid
expression. N. gonorrhoeae N313, a non-piliated strain
expressing the CEACAM-binding Opa57 (17), was used for all
experiments except for some experiments visualizing Akt-PH recruitment
in which strain N309 expressing the CEACAM-binding Opa52
was used instead. These gonococcal strains were generously provided by
Dr. T. F. Meyer (Max-Planck-Institut fur Infektionsbiologie,
Berlin, Germany). Bacteria were grown on GC agar plates supplemented
with 1% Isovitalex at 37 °C in 5% CO2.
Gentamicin Resistance Assays--
HeLa-CEACAM cells were grown
to 50-80% confluency in 24-well plates. Cells were washed three times
in serum-free RPMI 1640 medium prior to infection and incubated in
serum-free medium (with inhibitors as indicated) for Microscopic Analysis of Invasion--
For microscopy assays,
infections were performed as for the gentamicin assays with the
following exceptions. HeLa-CEACAM cells were grown on glass coverslips.
In experiments for scoring efficiency of uptake, as well as in some of
the experiments visualizing GFP probes, the cells were washed three
times with RPMI 1640 medium after centrifugation of bacteria to remove
loosely adherent bacteria. After centrifugation (and washing when it
was performed), cells were incubated with bacteria at 37 °C for
various times from 20 min to 3 h. Also a 10-fold lower number of
bacteria were added to HeLa-CEACAM1 cells in order to obtain levels of
bacterial binding to the cells comparable to that seen with the
HeLa-CEACAM3 cells. After infection the cells were washed two times
with phosphate-buffered saline + 1 mM Ca2+, 1 mM Mg2+ and then fixed in 4% paraformaldehyde.
Fixed coverslips were processed for microscopy as follows.
Extracellular bacteria were labeled by incubating with polyclonal
rabbit anti-gonococcal antibody UTR01 and then Alexa488-anti-rabbit
antibody (or, in experiments where GFP constructs were expressed,
Cy5-anti-rabbit). Cells were then permeabilized in 0.4% Triton X-100
for 15 min after which they were incubated with UTR01 as before and
then with Cy3-goat anti-rabbit; in experiments where scoring of
uptake was performed, Alexa633-phalloidin was also included at this
step to label actin to facilitate localization of the HeLa cells. Cells
were then washed three times in phosphate-buffered saline and mounted
using Dako fluorescence mounting medium. All incubations with
antibodies were preceded by three washes in phosphate-buffered saline
and a 10-min incubation in 0.2% bovine serum albumin to block, and all
antibody labeling steps were performed for 1 h at room temperature in the presence of 0.2% bovine serum albumin. For experiments with
LAMP-2 labeling, bacteria were detected either by prelabeling with FITC
or with UTR01 and Cy3-anti-rabbit, and LAMP-2 was detected with
antibody H4B4 and either Texas Red- or Alexa488-conjugated anti-mouse
secondary antibodies. Slides were analyzed by confocal microscopy using
a Zeiss LSM 510 laser-scanning confocal microscope (Carl Zeiss, Inc.)
with a 100× oil immersion objective and the conventional laser
excitation and filter sets or by fluorescence microscopy using a Leica
DMIRB/E microscope equipped with a Hamamatsu Orca ER camera controlled
by OpenLab v.2.2.5 digital imaging software (Improvision).
For experiments with live cells, bacteria were labeled with TRITC
before infection. Confocal stacks of z-slices were acquired at
successive time points during infection. Fluorescence intensity was
quantified by measuring the mean fluorescence intensity of the 50 brightest pixels in a region of interest containing the phagosome; this
was then divided by the average of the mean fluorescence intensities of
two separate reference regions of cytoplasm to correct for effects of photobleaching.
pH Measurements--
Gonococci were prelabeled by incubating in
12 µg/ml FITC in phosphate-buffered saline, pH 8.5, with gentle
shaking in the dark for 30 min. Bacteria were then washed in 1% bovine
serum albumin to quench residual FITC, and the bacteria were used to infect HeLa-CEACAM3 cells. After 30 min of infection, coverslips were
moved into a Leiden chamber and placed on a 37 °C heated stage of a
Leica DMIRB/E inverted fluorescence microscope. pH measurements were
performed as described previously (19). Briefly, images were acquired
using Metafluor software (Universal Imaging) with excitation at either
440 or 490 nm. pH was estimated from the ratio of fluorescence
intensity at 440 and 490 nm. For each field acquired, the pH of the
extracellular buffer was rapidly changed between 6.15 and 7.35;
internal bacteria could be identified retrospectively as those for
which the fluorescence intensities did not change significantly upon
change of the buffer. pH measurements were performed over a period of
20 min. Calibration was then performed in situ with
nigericin and buffers of defined pH values from 5.75 to 7.35. A minimum
of 50 bacteria was analyzed at each pH to construct the calibration
curves. For determining significance of differences of means,
two-tailed Student's t tests were used.
Inhibition of PI3K Increases Bacterial Recovery in Gentamicin
Assays--
We first assessed bacterial invasion of transfected
epithelial cells by means of standard gentamicin resistance assays.
Transfected HeLa cells stably expressing either CEACAM1 or CEACAM3 were
infected with the gonococcal strain N313 expressing the CEACAM-specific Opa57. After allowing for internalization, adherent
extracellular bacteria were killed by treatment with the
cell-impermeant antibiotic gentamicin. Host cellular membranes were
then solubilized to release internal bacteria that were plated on GC
agar, and the resulting colonies were counted. To obtain an indication
of whether PI 3-kinases are required for bacterial internalization,
uptake assays were also performed after pretreatment of the HeLa cells
with PI3K inhibitors.
Surprisingly we observed a substantial increase in the
number of N. gonorrhoeae colonies recovered after gentamicin
treatment when the HeLa-CEACAM3 cells were treated with the PI3K
inhibitor wortmannin (Fig.
1A). Similar results were
obtained with LY294002, a drug that inhibits PI3Ks by a distinct
mechanism (Fig. 1C). Wortmannin and LY294002 also typically
increased the recovery of gonococci following their infection of
HeLa-CEACAM1 cells, although the amplitude of the effects was somewhat
more variable (ranging from an insignificant to a 5-fold increase
observed in independent experiments). No difference in the number of
total (intracellular plus extracellular) bacteria associated with
either of the HeLa cell lines was observed between cells that were
untreated or treated with either inhibitor (Fig. 1, B and
D).
The unexpected finding that bacterial recovery was increased after
treatment with the inhibitors could be interpreted to indicate that
impairment of PI3K stimulates bacterial uptake. To test this notion and
to obtain an independent measure of bacterial internalization, we
directly observed bacterial invasion by fluorescence microscopy. CEACAM-transfected HeLa cells were infected and then fixed and processed for immunofluorescence. Extracellular and internal bacteria were distinguished by immunostaining with an anti-gonococcal antibody before and after permeabilization of the host cells, respectively. Notably, as illustrated in Fig. 2,
inhibition of PI 3-kinases did not increase the efficiency of uptake
mediated by either CEACAM1 or CEACAM3. In fact, for HeLa-CEACAM3 cells,
PI3K inhibition significantly lowered the proportion of associated
bacteria that became internalized (Fig. 2, p < 0.014).
The results in Figs. 1 and 2 indicate that PI3K inhibition leads to no
change or a decrease in bacterial uptake as judged by direct
visualization yet results in an increase in bacterial recovery in
gentamicin assays. While these results appear at first sight
contradictory, it should be noted that gentamicin resistance assays
reflect a combination of two factors. For bacteria to be recovered as
colony-forming units, they must not only be internalized by the
eukaryotic cell to avoid contact with gentamicin, but they must also
survive in the intracellular milieu until the time of host cell lysis.
In this regard, it is noteworthy that a sizable fraction of the
bacteria associated with the HeLa cells is seen to be internalized by
microscopy (Fig. 2), but only a small fraction of the total
cell-associated bacteria is recoverable as gentamicin-resistant colony-forming units at longer times (compare scales of
axes in Fig. 1, A and C versus
B and D). This implies that extensive death of
intracellular gonococci is occurring over time. We hypothesized that
the divergent observations made using the two assay systems result from
PI 3-kinases being involved in multiple steps during bacterial
internalization and killing. PI3K activity appears to be required for
optimal initial uptake of bacteria via CEACAM3. However, in the absence
of PI3K activity, survival of those bacteria that do become
internalized via either CEACAM receptor must be enhanced, leading to a
greater recovery of gentamicin-resistant bacteria.
Phosphatidylinositol 3,4,5-Trisphosphate (PIP3)
Accumulates Locally during CEACAM3-mediated Uptake--
In further
considering possible roles for PI 3-kinases during bacterial invasion,
it is important to note that several classes of PI3Ks are inhibited by
LY294002 and wortmannin, and the activity of these different enzymes
can result in synthesis of distinct 3-phosphoinositides. Class I PI3Ks
in vivo use mainly phosphatidylinositol 4,5-bisphosphate as
a substrate to generate PIP3, whereas class III PI3Ks
generate predominantly phosphatidylinositol 3-phosphate (PI3P) using
phosphatidylinositol as a substrate (20). These phosphoinositide
species perform distinct functions in cell signaling and may be
differentially involved in bacterial invasion and phagosome maturation.
A major advance in studies of phosphoinositide metabolism has been the
recent development of probes consisting of GFP fused to protein domains
that bind with high affinity to specific phosphoinositides (21). Using
such probes, localized accumulation of specific phosphoinositide second
messengers can be evaluated with high spatial and temporal resolution.
It was thus demonstrated that engagement of Fc
Accumulation of Akt-PH-GFP in HeLa-CEACAM3 cells was rarely seen on the
membrane of phagosomes containing fully internalized bacteria. The
paucity of Akt-PH-GFP in sealed phagosomes suggests that
PIP3 accumulation is a transient phenomenon during
bacterial uptake. To better appreciate the kinetics of PIP3
accumulation, the dynamics of Akt-PH-GFP localization were followed in
living HeLa-CEACAM3 cells during bacterial infection (Fig.
4). Accumulation of the probe occurred
while bacteria were bound to the cell surface. The intensity peaked at
roughly the time of phagosomal closure as estimated from
reconstructions in the xz and yz planes of stacks of confocal images
(data not shown). Following internalization of the bacteria, the
fluorescence intensity on the formed phagosome rapidly declined until
no enhancement over the cytoplasmic intensity could be observed.
Quantitation of a representative experiment is shown in Fig.
4G. Thus, localized PIP3 accumulation is an
early and transient event during CEACAM3-mediated uptake. This
observation, along with the inhibitory effects of wortmannin on
bacterial uptake via CEACAM3, suggests a functional role for this
PIP3 formation in CEACAM3-mediated internalization of
bacteria.
PI3P Accumulates in Phagosomes Containing Gonococci--
As
discussed above, the enhanced recovery of gentamicin-resistant bacteria
after inhibition of PI3K is not due to an increase in internalization,
implying an increase in bacterial survival inside the cells. The early
and transient appearance of PIP3 during uptake mediated by
CEACAM3, but not CEACAM1, makes it unlikely that this
3-phosphoinositide is regulating bacterial survival within the cell,
which was enhanced by PI3K inhibitors in both CEACAM1- and
CEACAM3-expressing cells. An alternate PI3K product that may be
involved in regulating bacterial survival after internalization is the
singly phosphorylated PI3P. This lipid is produced in vivo primarily through action of class III PI 3-kinases and is localized predominantly in endosomal compartments (6). To test the possible involvement of PI3P in bacterial invasion and intracellular traffic, two probes that specifically bind to this phospholipid were used. These
probes consist of GFP fused to either the PX domain of the p40phox subunit of the NADPH oxidase (phox-GFP) or to
two tandem repeats of the FYVE domain from EEA1 (early endosome
autoantigen 1; 2-FYVE-GFP). Both have been used previously as probes of
PI3P localization (15, 16). First HeLa-CEACAM3 cells were transiently
transfected with phox-GFP. In uninfected cells, the probe localizes to
the cytoplasm as well as to small vesicular structures, reflecting the
existence of PI3P in endosomal compartments. These cells were then
infected with gonococci. In contrast to the early, transient accumulation of Akt-PH-GFP, phox-GFP was primarily seen on formed phagosomes (Fig. 5,
A-C). On occasion, it did appear to localize also to phagosomes that had not yet closed in which bacteria were still
accessible to labeling with external antibodies; however, such
recruitment occurred only occasionally and was less intense (data not
shown). In contrast, virtually all intracellular bacteria were found to
be in phagosomes that accumulated phox-GFP (Fig. 5,
A-C). The acquisition of phox-GFP appeared to be
long-lived as phagosomes were still labeled with the probe several
hours after infection (data not shown). As expected, phox-GFP
localization to phagosomes was abolished by wortmannin treatment (Fig.
5, D-F). Similar persistent accumulation on
gonococcal phagosomes was seen for the 2-FYVE-GFP probe, verifying the
presence of PI3P (Fig. 5, G-I). We further
examined the kinetics of PI3P accumulation by following the phox-GFP
probe during infection in living HeLa-CEACAM3 cells (Fig.
6). These experiments indicated that
phox-GFP probe was rapidly acquired shortly after the apparent closure
of the phagosome and persisted for long periods of time (>1 h).
In infected HeLa-CEACAM1 cells, phox-GFP and 2-FYVE-GFP were also found
to accumulate on gonococcal phagosomes (Fig. 5,
J-L, and data not shown). Unlike phagosomes
formed by CEACAM3-expressing cells, which were uniformly labeled, only
some of the apparently internalized bacteria were positive for the
probes in the HeLa-CEACAM1 cells. It is unclear whether this reflects
the existence of two subpopulations of CEACAM1 phagosomes or a
heterogeneous kinetics of acquisition and/or loss of PI3P.
PI3K Activity Is Required for Phagosomal Maturation--
The
appearance of PI3P on the bacteria-containing phagosome suggests that
this phosphoinositide plays a role in the maturation process that
confers microbicidal properties to the phagosome. To test whether
inhibition of class III PI3Ks affects phagosomal maturation, we
measured the acquisition by phagosomes of the late endosomal and
lysosomal marker LAMP-2. While LAMP-1 is cleaved by the IgA1 protease
that is secreted by N. gonorrhoeae, LAMP-2 is not targeted
by this enzyme and thus remains a reliable marker (23, 24). An hour
after infection, the majority of phagosomes in HeLa-CEACAM3 cells are
positive for LAMP-2, indicating fusion with endosomal compartments
(Fig. 7, A and B).
Remarkably, in cells treated with PI3K inhibitors, acquisition of this
marker is substantially reduced (Fig. 7,
C-E).
While LAMP-2 acquisition provides an indication of the extent of fusion
of the phagosome with late endosomal/lysosomal compartments, it is
unclear whether this protein plays any role in bacterial killing. We
therefore sought to obtain a separate functional measure of the killing
capacity of the phagosomes containing gonococci. An important aspect of
phagosomal maturation in professional phagocytes that is believed to
contribute to the bacteriostatic or bactericidal character of the
phagosome is the acidification of its lumen. Thus, we investigated
whether acidification of the gonococci-containing phagosome is a
PI3K-dependent process. Gonococci were covalently labeled
with a pH-sensitive dye and allowed to invade HeLa-CEACAM3 cells. The
pH of phagosomes containing internalized bacteria was then determined
by ratiometric imaging of bacterial fluorescence inside live HeLa
cells. In control cells, after 30 min of internalization bacteria were
found to reside in an acidified compartment with a mean pH of 5.85 (Fig. 8A). In
wortmannin-treated cells, phagosomes also acidified, but the
acidification was significantly attenuated (mean pH value of 6.18, p < 3.5 × 10
These results raised the possibility that the inability of
phagosomes to acidify fully contributes to the increased
gonococcal survival in cells treated with PI3K inhibitors. We therefore
tested whether direct inhibition of acidification could similarly
promote intracellular survival. To this end, cells were treated with
concanamycin A, an inhibitor of the vacuolar
(H+)- ATPases. As illustrated in Fig. 8, the number of viable
bacteria recovered in gentamicin resistance assays was significantly
enhanced upon drug treatment. Microscopic analysis of invasion
confirmed that concanamycin A treatment does not enhance
internalization per se (data not shown), indicating that the
increased bacterial recovery must have resulted from improved
intracellular survival. Consistent with the model that inhibition of PI
3-kinase activity favors gonococcal survival by impairing acidification
of the bacteria-containing phagosome, we found that the effects of
LY294002 and concanamycin A on bacterial recovery were not additive
(Fig. 8B). In fact, treatment with a combination of LY294002
and concanamycin A led to a lower recovery of bacteria than that in
cells treated with concanamycin A alone, as would be expected if
LY294002 inhibits bacterial internalization via CEACAM3, while having
little or no positive effect on survival beyond that already achieved
with concanamycin A. Thus, PI3K-dependent phagosomal
acidification appears to be an important factor in determining the
extent of gonococcal killing by the host cells.
Our results reveal distinct roles for two types of PI3K during
CEACAM-mediated internalization and elimination of N. gonorrhoeae by human cells. PIP3 locally accumulates
during uptake via CEACAM3 but not CEACAM1. This accumulation is
reminiscent of that seen during Fc Gentamicin resistance assays are routinely used to measure bacterial
internalization into mammalian cells. The discrepancies we observed
between invasion measured by such assays and that measured by direct
visualization underscore the fact that gentamicin assays must be
interpreted cautiously as they require intracellular survival of the
bacteria for final detection. This caveat is particularly important in
cases like that of gonococci in our system in which extensive
intracellular death of bacteria is apparent.
Little is known about the intracellular life of gonococci. They are
capable of performing Opa-dependent transcytosis across epithelial cell layers (26), a process that is presumably important in
bacterial colonization of the submucosa (27). Transepithelial migration
occurs through an intracellular route and takes many hours to occur
(26). Clearly then at least some bacteria must survive sufficiently
long periods to emerge from the basolateral side of the epithelial
cells. In the HeLa-CEACAM cells, bacteria do not appear to replicate in
the intracellular compartment; rather the number of viable
intracellular bacteria steadily declines over several hours (data not
shown). This stands in contrast to bacteria that actively replicate
within either epithelial or phagocytic cells, such as
Salmonella spp. It should be noted, however, that prolonged
survival of even a low percentage of internalized gonococci may be a
crucial factor in determining the outcome of infection. Therefore, it
is important to understand the factors that influence the intracellular
fate of these bacteria. Our results imply that PI3K activity is an
important factor in the maturation of the gonococcal phagosome, most
likely by virtue of its generation of PI3P on phagosomal membranes. The
role of PI 3-kinases in affecting intracellular survival of other
pathogens is poorly understood. Mycobacteria promote their survival
within macrophages by arresting phagosome maturation at an early stage,
and this may be attributed to inhibition of class III PI3Ks by
mycobacterial products (28). However, the role of PI3K in mycobacterial
survival has not been directly investigated. The role of PI3K in
intracellular survival of Salmonella is controversial. In
one report, inhibition of PI3K had no effect on intracellular
replication of bacteria (29), whereas in another report, replication of
Salmonella was enhanced when assessed following several
hours after internalization (30). Thus, further analysis of the
role of phosphoinositides in survival of intracellular bacteria will be important.
A striking finding in our study was the persistence of PI3P in the
membrane of phagosomes formed upon ingestion of gonococci by
HeLa-CEACAM3 cells. PI3P accumulated on the phagosomes for at least
several hours after bacterial uptake. This observation stands in sharp
contrast to the results reported for phagosomes formed by Fc
receptor-mediated phagocytosis or for Salmonella-containing vacuoles formed in epithelial cells. In both of these cases, PI3P accumulated only transiently, largely disappearing within 30 min (16,
29, 31, 32). This suggests basic differences in the process of
maturation of the gonococcal phagosomes. These differences may reflect
interference with phagosomal maturation induced by bacterial factors,
e.g. the porin PorB (33) or the secreted endopeptidase that
cleaves LAMP-1 (24), or may be due to factors specific to signaling
through CEACAM receptors. Studies to address these issues will help to
reveal how such factors influence the intracellular processing of this
important pathogen.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
30 min.
Gonococci were scraped from a plate after overnight growth, resuspended
in RPMI 1640 medium, washed once, and resuspended to a concentration of
2 × 107/ml. Gonococci were added to the cells at a
multiplicity of infection of ~50:1 (0.5 ml/well in 24-well plates).
Samples were subjected to centrifugation for 5 min at 67 × g to bring the bacteria into association with the HeLa
cells. Cells were then incubated in RPMI 1640 medium at 37 °C in 5%
CO2 for 3 h, and then gentamicin (100 µg/ml) was
added for 2 h at 37 °C to kill extracellular bacteria. Wells
were washed, and saponin was added to permeabilize cells followed by
plating of appropriate dilutions of the lysate on GC agar (18). For
measuring total bacterial association, lysis with saponin was done
without prior gentamicin treatment.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
Inhibition of PI 3-kinases causes enhanced
recovery of bacteria in gentamicin resistance assays. Gentamicin
resistance assays were performed with HeLa-CEACAM1 or HeLa-CEACAM3
cells as described under "Experimental Procedures."
A and B, infection of HeLa-CEACAM1 or
HeLa-CEACAM3 cells was performed with (black bars) or
without (white bars) treatment of cells with 100 nM wortmannin. C and D,
infection was performed with (black bars) or without
(white bars) treatment of cells with 50 µM
LY294002. Means ± S.E. of triplicate samples from individual
experiments are shown. Similar effects of PI3K inhibition were seen in
four independent experiments. cfu, colony-forming
units.
View larger version (12K):
[in a new window]
Fig. 2.
Inhibition of PI 3-kinases does not stimulate
bacterial uptake. HeLa-CEACAM1 or HeLa-CEACAM3 cells were infected
with gonococci for 30 min. Bars indicate the fraction of
total associated bacteria that are internalized. White bars
show untreated controls; black bars show cells treated with
100 nM wortmannin. Results for HeLa-CEACAM1 and
HeLa-CEACAM3 were from separate experiments performed in triplicate
(CEACAM1) or quadruplicate (CEACAM3); bars show means ± S.E. with >30 cells counted in each sample. The number of total
associated bacteria per cell is as follows: CEACAM1 control, 12 ± 2; CEACAM1 treated with wortmannin, 11 ± 2; CEACAM3 control,
11 ± 3; CEACAM3 with wortmannin, 17 ± 6. Similar results
were obtained for CEACAM3 with 100 µM LY294002.
receptors can lead to
highly localized accumulation of PIP3, presumably via
activation of a class I PI3K (22). CEACAM3, but not CEACAM1, contains
an ITAM similar to those found in immune receptors like the
Fc
receptor. It was therefore conceivable that a class I PI3K
might be differentially involved in uptake mediated by these two
distinct CEACAM receptors. To analyze this possibility, HeLa cells
expressing CEACAM1 or CEACAM3 were transiently transfected with a
plasmid expressing the PH domain of Akt, which binds with high affinity
to PIP3, fused to GFP (Akt-PH-GFP). The Akt-PH-GFP probe
localizes to the cytoplasm and nucleus in uninfected cells (data not
shown). However, upon infection of HeLa-CEACAM3 cells with gonococci,
we observed a striking accumulation of the probe at sites where
bacteria bound to the cell surface (Fig. 3, A and B).
Akt-PH-GFP was particularly concentrated in pseudopod-like extensions
of the plasma membrane extending around the bacteria (Fig.
3A, inset). The accumulation of Akt-PH-GFP was
abolished upon inhibition of PI3K by wortmannin (Fig. 3, C
and D), confirming that the probe reports the distribution
of 3-polyphosphoinositides. The main product of PI3K sensed by the
probe is likely PIP3, although phosphatidylinositol
3,4-bisphosphate, which binds to the probe in vitro,
may contribute to the signal. Notably, in contrast to the observations
in HeLa-CEACAM3 cells, no noticeable accumulation of Akt-PH-GFP at
sites of bacterial entry was observed during invasion of HeLa-CEACAM1
cells (Fig. 3, E and F). Thus, activation of
localized synthesis of PIP3 is specific to the
ITAM-containing CEACAM3 receptor.
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Fig. 3.
Localized accumulation of Akt-PH-GFP during
CEACAM3-mediated uptake. HeLa-CEACAM3 (A-D)
or HeLa-CEACAM1 (E and F) cells expressing
Akt-PH-GFP were infected with gonococci for 20-30 min. In A
and B, gonococcal strain N309 was used; similar
accumulations of Akt-PH-GFP were obtained with strain N313.
A, C, and E show Akt-PH-GFP
fluorescence. B, D, and F show
labeling of bacteria with anti-gonococcal antibody. Arrows
in A and B indicate sites of Akt-PH-GFP
accumulation and corresponding bound bacteria, respectively.
Inset in A and B shows a higher
magnification of a region showing accumulation of Akt-PH-GFP in
pseudopods extending around a bacterium. In C and
D, cells were treated with 100 nM wortmannin.
Bars, 5 µm.
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Fig. 4.
Time course of Akt-PH-GFP accumulation during
CEACAM3-mediated uptake. Uptake of TRITC-conjugated bacteria by
HeLa-CEACAM3 cells expressing Akt-PH-GFP was followed over time by
confocal microscopy. A and C-F,
Akt-PH-GFP fluorescence. B, bacteria.
C-F show a higher magnification with
D corresponding to the time shown in A. Bar in A, 5 µm. Bar in C,
1 µm. G, fluorescence intensity of the illustrated
phagosome was quantitated as described under "Experimental
Procedures." Letters indicate times corresponding to the
images in C-F.
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Fig. 5.
PI3P accumulates on gonococcal
phagosomes. HeLa-CEACAM3 (A-I) or
HeLa-CEACAM1 (J-L) cells were transfected with
phox-GFP (A-F and J-L) or
2-FYVE GFP (G-I) and infected with gonococci for
60-75 min (A-F) or 30 min
(G-L). A, D, G,
and J show GFP fluorescence. B, E,
H, and K show total bacteria. C,
F, I, and L show external bacteria. In
D-F, cells were treated with 100 nM wortmannin. Arrows indicate internalized
bacteria that do not label with phox-GFP after wortmannin treatment.
Bars, 5 µm.
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Fig. 6.
Time course of phox-GFP accumulation during
CEACAM3-mediated uptake. Uptake of TRITC-conjugated bacteria by
HeLa-CEACAM3 cells expressing phox-GFP was followed over time by
confocal microscopy. A and C-F,
phox-GFP fluorescence. B, bacteria.
C-F show a higher magnification with
D corresponding to the time shown in A. The
arrow indicates the position of a bacterium. G,
fluorescence intensity of the illustrated phagosome was quantitated as
described under "Experimental Procedures." Letters
indicate times corresponding to images in C-F.
Bar in A, 5 µm. Bar in C,
1 µm.
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Fig. 7.
Inhibition of PI3K impairs phagosomal
acquisition of LAMP-2. HeLa-CEACAM3 cells were infected with
gonococci in the absence (A and B) or presence
(C and D) of 100 nM wortmannin,
incubated for 1 h, and processed for detection of LAMP-2 by
immunofluorescence microscopy. A and C show
LAMP-2 immunofluorescence. B and D show bacteria.
All illustrated bacteria are intracellular. Bars, 5 µm.
E, in a separate experiment, phagosomal acquisition of
LAMP-2 marker 1 h after infection with or without treatment of
HeLa-CEACAM3 cells with 50 µM LY294002 was quantified
(n = 645 for control, n = 399 for
+LY294002).
6). Thus, PI3K
activity is required for optimal acidification of the gonococcal
phagosome.
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Fig. 8.
Acidification of the gonococcal
phagosome. A, HeLa-CEACAM3 cells were infected for 30 min with FITC-labeled gonococci in the absence or presence of 100 nM wortmannin, and the pH of internalized bacteria was
measured as described under "Experimental Procedures." White
circles, control; black squares, wortmannin-treated.
Each symbol represents the mean pH ± S.E. from an
independent experiment, each measuring separately the pH of a minimum
of 28 internalized bacteria. B, inhibition of endosomal
acidification enhances survival of gonococci. HeLa-CEACAM3 cells were
infected with gonococci, and gentamicin resistance assays were
performed as in Fig. 1 in the absence or presence of 50 µM LY294002, 200 nM concanamycin A, or both
together as indicated. White bars indicate total bacterial
association (left axis); black bars
indicate viable bacteria recovered after gentamicin treatment
(right axis). Means ± S.E. of triplicate samples are
shown; similar results were obtained in two separate experiments.
LY, LY294002; con.A, concanamycin A;
cfu, colony-forming units.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor-mediated phagocytosis by
macrophages (22). CEACAM3, but not CEACAM1, contains in its cytoplasmic
domain an ITAM, similar to those found in Fc receptors, that is
involved in signaling cytoskeletal changes underlying CEACAM3-mediated uptake (4, 25). Our results demonstrate that binding of Opa-expressing bacteria to CEACAM3 receptors also induces localized recruitment and/or
activation of a class I PI 3-kinase and suggest that the activity of
this kinase is required for optimal uptake. Inhibition of PI3K does
not, however, lead to a complete block in uptake of gonococci. Previous
experiments with Fc
receptor-mediated engulfment demonstrated that
the dependence of phagocytosis on PI3K activity was not absolute,
decreasing in direct proportion with the size of the particles being
engulfed (12). Interestingly when IgG-opsonized beads of 1-µm
diameter, the approximate size of gonococci, were used, Fc
receptor-mediated phagocytosis was also only partially inhibited
by blockade of PI3K (12) as seen here for CEACAM3-mediated uptake of bacteria.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Keith Ireton for helpful advice throughout this work and Drs. Michael Yaffe and Tobias Meyer for kindly providing the phox-GFP and Akt-GFP constructs.
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FOOTNOTES |
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* This research was funded in part by the Canadian Institutes of Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Supported by a fellowship from the Canadian Cystic Fibrosis Foundation.
Current holder of the Pitblado Chair in Cell Biology at the
Hospital for Sick Children.
§§ Supported by a New Investigator Award from the Canadian Institutes of Health Research. To whom correspondence should be addressed: Dept. of Medical Genetics and Microbiology, University of Toronto, 4381 Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-946-5307; Fax: 416-978-6885; E-mail: scott.gray.owen@utoronto.ca.
Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M211879200
2 S. E. McCaw, J. Schneider, E. H. Liao, W. Zimmermann, and S. D. Gray-Owen, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: Opa, opacity-associated; CEACAM, carcinoembryonic antigen-related cellular adhesion molecule; PI, phosphatidylinositol; PI3K, phosphatidylinositol 3-kinase; PH, pleckstrin homology; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PI3P, phosphatidylinositol 3-phosphate; GFP, green fluorescent protein; LAMP-1, lysosome-associated membrane protein 1; LAMP-2, lysosome-associated membrane protein 2; ITAM, immunoreceptor tyrosine-based activation motif.
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