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Infection and Immunity, October 2000, p. 5735-5741, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Activation of Host Phospholipases C and D in
Macrophages after Infection with Listeria
monocytogenes
Howard
Goldfine,*
Sandra J.
Wadsworth, and
Norah C.
Johnston
Department of Microbiology, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076
Received 11 May 2000/Returned for modification 19 June
2000/Accepted 11 July 2000
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ABSTRACT |
Infection of the J774 murine macrophage-derived cell line with
Listeria monocytogenes results in several elevations of
intracellular calcium during the first 15 min of infection. These
appear to result from the actions of secreted bacterial proteins,
including phosphatidylinositol-specific phospholipase C (PI-PLC), a
broad-range phospholipase C, and listeriolysin O (LLO) (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770-1778, 1999). We have
measured hydrolysis of host PI and the activation of host
polyphosphoinositide-specific PLC and host phospholipase D (PLD) during
infection with wild-type and mutant L. monocytogenes.
Elevated hydrolysis of host PI occurred within the first 10 min of
infection and was dependent on both bacterial PI-PLC and LLO, both of
which were required for the earliest elevations of intracellular
calcium in the host cell. A more rapid hydrolysis of host PI was
observed at 30 min after infection, at the time when wild-type bacteria
have been internalized. Activation of host PLC, also occurred in the
first 10 min of infection but was not dependent on the presence of
bacterial PI-PLC. Similar observations were made in murine bone
marrow-derived macrophages. In J774 cells, activation of host PLD was
observed after 20 min of infection and was dependent on bacterial LLO.
Mutants in the bacterial phospholipases produced levels of PLD
activation similar to those produced by the wild type. Phorbol
myristate acetate (PMA) also activated host PLD, while long-term
treatment with PMA resulted in loss of the ability of L. monocytogenes to activate host PLD, suggesting an involvement of
protein kinase C (PKC) in the activation of PLD. Rottlerin, an
inhibitor of PKC 
in J774 cells, also inhibited the activation of
PLD, but hispidin, an inhibitor of PKC 
I and II, did not.
Pretreatment of J774 cells with the PLD inhibitor,
2,3-diphosphoglycerate partially inhibited escape of the bacteria from
the primary phagocytic vacuole.
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INTRODUCTION |
The earliest events in the
interaction of Listeria monocytogenes with mammalian cells
appear to involve the activities of bacterial secreted proteins before
internalization of these bacteria. On infection of the J774 murine
macrophage cell line, these activities delay uptake of wild-type
bacteria into the phagosome (36). Subsequent growth in the
cytoplasm and cell-to-cell spread are completely dependent on the
ability of the bacterium to mediate escape from a vacuole (12, 26,
35). Two genes, hly and plcA, in a cluster
of six genes on the bacterial chromosome have been implicated in escape
from the primary vacuole of a macrophage. They encode listeriolysin O
(LLO) and a phosphatidylinositol-specific phospholipase C (PI-PLC),
respectively. A third gene, prfA, which is adjacent to
plcA, encodes a positive regulatory protein, PrfA, which is
required for the induction of all genes in this virulence cluster
(25, 29). LLO has been shown to be absolutely required for
escape from the primary vacuole of a macrophage (12, 26) and
for mouse virulence (9, 13, 18, 26, 29). Assays for escape
from the primary vacuole show that mutants in PI-PLC are between 30 and
65% less likely to be found in the cytoplasm of a bone marrow-derived
macrophage than a wild-type strain at 1.5 h postinfection
(7, 33). These mutants show reduced growth compared to the
wild type in mouse liver; however, the mouse 50% lethal dose is
only slightly increased upon intravenous infection (7).
PI-PLC of L. monocytogenes is a member of a family of
homologous enzymes secreted by gram-positive bacteria. Like other
bacterial PI-PLCs, the enzyme from L. monocytogenes has high
specificity for PI with no detectable activity on PI-4-P or
PI-4,5-P2, eukaryotic lipids involved in intracellular
signaling. It has relatively low activity on glycosyl-PI-anchored
eukaryotic membrane proteins, which are actively cleaved by other
bacterial PI-PLCs (14, 16).
The ability of L. monocytogenes to escape from a phagosome,
grow in the cytoplasm, and spread from cell to cell is essential for
the pathogenesis of this food-borne, human and animal pathogen. In
humans, infections with L. monocytogenes tend to occur
in immunocompromised adults, pregnant women, and the elderly.
They can produce septic abortions of the fetus and meningoencephalitis
and are often fatal (11, 28).
Since bacterial LLO and PI-PLC activities appear to be important for
elevation of intracellular Ca2+ in host cells
(36), it seemed possible that there is a connection between
escape from the vacuole and activation of certain host cell functions
that are dependent on elevated intracellular Ca2+. Among
these is the activation of host PLC isoforms, which hydrolyze PI-4-P
and PI-4,5-P2 (27, 31). The hydrolysis of host
phosphoinositides by bacterial and host PLCs also results in the
formation of diacylglycerol (DAG), which is an activator of eukaryotic
protein kinase C (PKC) isoforms (24). Activation of the
classical isoforms of PKC also requires elevated intracellular
Ca2+. Since PKC has been implicated in activation of
phospholipase D (PLD) (31) and PLD influences the
internalization of another facultative intracellular pathogen,
Mycobacterium tuberculosis (20), we have also
examined the activation of this host function in infected J774 cells.
Our studies show that there is an LLO- and a PI-PLC-dependent
hydrolysis of host PI in J774 cells. Activation of J774 cell
polyphosphoinositide PLC and PLD was also observed, and these
activities were completely dependent on the expression of
bacterial LLO.
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MATERIALS AND METHODS |
Bacterial strains and mammalian cells.
The wild-type
L. monocytogenes strain used in this study was 10403S,
belonging to serotype 1 (4). The mutant strains derived from
strain 10403S were strain DP-L2161 (
hly) (17),
strain DP-L1552 (plcA) (7), strain DP-L1935
(plcB), and strain DP-L1936 (plcA plcB)
(33). These strains and the J774 murine macrophage cell line
were obtained from Daniel A. Portnoy (University of California,
Berkeley) and grown as described previously (36). Murine
bone marrow macrophages and J774 cells were propagated as described by
Portnoy et al. (26), except that bone marrow macrophages
were routinely grown in 60-mm petri dishes (Lab-Tek; American
Scientific Products, McGaw Park, Ill.).
Infection of cells prelabeled with
[2-3H]inositol.
Bone marrow-derived macrophages and
J774 cells were plated in six-well dishes at 1.3 × 106 or 1.0 × 106 cells/well,
respectively. After overnight growth in 1.5 ml of Dulbecco modified
Eagle medium (DMEM) minus inositol containing 10% fetal bovine serum
(FBS) and 10 µCi of myo-[2-3H]inositol
(Amersham) per well, the medium was removed, and 2 ml of prewarmed DMEM
plus 10% FBS was added. After 10 min at 37°C, 20 µl of 1 M LiCl
was added to each well. A suspension of L. monocytogenes was
prepared by inoculation of 0.5 ml of an overnight culture grown in
brain heart infusion (BHI) broth into 3.5 ml of fresh BHI broth
followed by growth on a rotator at 37°C for 2 h. After centrifugation of 1 ml of this logarithmic-phase culture in a microcentrifuge for 1 min and washing with 1 ml of phosphate-buffered saline (PBS), the bacteria were suspended in PBS to provide a density,
i.e., A620, of 1.2. After a further 10 min at
37°C in the CO2 incubator, the monolayer of cells was
infected with 60 µl of the bacterial suspension. Control cultures
received an equal volume of PBS. For incubations longer than 30 min,
the medium was completely removed at 30 min and replaced with fresh
medium containing 10 mM LiCl, and gentamicin was added to a final
concentration of 50 µg/ml. To harvest the cells at the times
indicated, the dishes were placed on ice; after removal of the medium,
0.5 ml of ice-cold 4% perchloric acid was added, and the cells were
scraped and transferred to screw-cap Eppendorf microcentrifuge tubes. The wells were scraped twice more with 0.2 ml of cold 4% perchloric acid, and the washes were combined with the first extract. The cell
suspensions were centrifuged at 2,000 rpm at 4°C. The supernatants from two parallel cultures were combined, diluted to 5 ml with deionized water, and neutralized to pH 7.5 to 8.0 with KOH solutions. The precipitate was centrifuged for 5 min at 2,000 rpm. The
supernatants were transferred to fresh tubes and diluted to 10 ml.
Carrier inositol-1-P (IP), inositol-1,4-P2
(IP2), and inositol-1,4,5-P3 (IP3),
10 µg of each, were added.
Separation of IP and polyphosphoinositide hydrolysis
products.
IP, IP2, and IP3 were separated
on 1-ml columns of Dowex AG1X8, 200-400 mesh-formate form as described
by Berridge et al. (3) and as modified by Watson
(38). Then, 2-ml fractions were collected, and tritium was
measured by liquid scintillation spectrometry. Chromatography of
radioactive standards of IP2 and IP3 was used
to verify the expected elution and separation of these compounds.
Assay of PLD.
J774 cells were grown in Iscove modified
Dulbecco medium (IMDM; BioWhittaker) supplemented with 2 mM glutamine
and 100 µg of nonessential amino acids (Gibco-BRL) per ml
(1). The cells were grown overnight in six-well plates at
106 cells/well in 2 ml of supplemented IMDM plus 10% FBS,
and the lipids were labeled with 5 µCi of
[9,10-3H]palmitic acid (Amersham) per well. One hour
before infection, the medium was removed, the cells were washed with
prewarmed PBS, and 2 ml of supplemented IMDM plus 1 mg of bovine serum
albumin BSA (fatty acid-free; Sigma) per ml was added. At 5 min before infection, ethanol at 20 µl per well was added. A 60-µl suspension of the appropriate L. monocytogenes strain, prepared as
described above, was added, and the cells were returned to the incubator.
The infection was stopped by removing the medium, chilling the plates
on ice, and washing the cells twice with PBS at 0°C. Then, 1%
methanolic HCl at 0°C (0.75 ml/well) was added, and the cells were
scraped off and transferred to 13-by-100-mm glass tubes; any remaining
cells were transferred twice with 0.3 ml of 1% methanolic HCl.
Carriers, 10 µg each of phosphatidic acid (PA) and
phosphatidylethanol (PEt), were added, and the cells were extracted by
the method of Bligh and Dyer (5). The lipid solutions were
evaporated to dryness and dried in a vacuum desiccator.
The lipids were chromatographed on Whatman LKD 60 thin-layer plates in
the upper phase of the solvent ethyl
acetate-
iso-octane-acetic
acid-water (13:2:3:10
[vol/vol/vol/vol]) (
1) and visualized
by spraying them
with 0.001% Primulin (Sigma) in acetone-H
2O (4:1).
The PA
and PEt bands were scraped into liquid scintillation vials
for
radioassay. The amount of PEt formed is expressed as the percent
counts
per minute (cpm) relative to total lipid counts per
minute.
Measurement of escape from the primary vacuole.
Escape of
L. monocytogenes from the primary phagocytic vacuole was
measured by labeling bacteria with fluorescien isothiocyanate (FITC)
and rhodamine-phalloidin as previously described (21, 36).
Briefly, cells which had been labeled with FITC prior to infection were
labeled with rhodamine-phalloidin after infection. Phalloidin binds to
polymerized actin, which is only associated with bacteria in the cytosol.
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RESULTS |
Formation of inositol-P upon infection of J774 cells with L. monocytogenes.
When J774 cells were infected with washed L. monocytogenes, there was a significant increase in
[3H]IP within 10 min of infection (P < 0.0001, n = 3). However, a much more rapid release was
observed at 30 min after infection (Fig.
1A and data not shown). The release of
[3H]IP was greatly diminished upon infection with mutants
with deletions in hly or plcA, the genes for LLO
and PI-PLC, respectively (Fig. 1B and 2). A mutant with a deletion in
plcB, the gene for the broad-range PLC (BR-PLC), produced
the same amount of [3H]IP as infection with the wild
type, and a mutant with deletions in both plcA and
plcB gave the same low level of [3H]IP as the
mutant in plcA alone (Fig. 2).
Thus, the observed hydrolysis of phosphoinositide was dependent on the
ability of L. monocytogenes to produce LLO and PI-PLC during
these early stages of infection. These requirements are consistent with
a need for both LLO and PI-PLC for elevation of intracellular calcium in infected J774 cells (36) and for the activation of PKC
and II isoforms (S. J. Wadsworth and H. Goldfine,
unpublished data).
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FIG. 1.
Formation of [3H]IP, IP2, and
IP3 in J774 cells prelabeled with
[3H]inositol after infection with L. monocytogenes. Radioactive products of PI (inositol-P), PIP
(IP2), and PIP2 (IP3) hydrolysis
were extracted from infected cells at the times indicated and separated
by ion-exchange chromatography as described in the text. (A and B)
[3H]IP after infection with the strains indicated. (C and
D) [3H]IP2 ( ) and
[3H]IP3 ( ) after infection with the
plcA strain (dashed lines) and hly strain
(dotted lines). Data for the wild type represent the mean ± the
standard deviation (SD) (n = 3) from a representative
experiment, and data for the mutant strains represent the mean ± the standard error of the mean (SEM) (n = 2) from a
representative experiment.
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FIG. 2.
Formation of [3H]IP in J774 cells
prelabeled with [3H]inositol after infection with
L. monocytogenes strains. IP was extracted from infected
cells at 30 min after infection and separated by ion-exchange
chromatography as described in the text. The data represent the
means ± the SEM for wild-type (n = 12), plcA
(n = 4), hly (n = 3), plcB (n = 5), and
plcA plcB (n = 4) strains. Differences
between wild-type and the hly, plcA, and
plcA plcB strains were all significant (P < 0.01, Mann-Whitney).
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Activation of host polyphosphoinositide-PLC during infection of
J774 cells with L. monocytogenes.
Mammalian isoforms of PLC
preferentially hydrolyze PI-4,5-P2 and PI-4-P to give rise
to IP3 and IP2, respectively (31). Elevated formation of [3H]IP3 and
[3H]IP2 was readily observed at 30 min and
was variably observed at 10 min after infection with the wild type
(Fig. 1C). A similar increase in these tritiated products was not
observed upon infection with a mutant in LLO and was lower with the
mutant in bacterial PI-PLC, but the latter did not meet a test of
statistical significance (Fig. 1D and 3).
A BR-PLC deletion mutant activated host PLC to the same extent as the
wild-type as did, surprisingly, the double mutant, plcA
plcB (Fig. 3). Thus, the broad-range phospholipase of L. monocytogenes is not required for activation of host PLC nor,
apparently, is activity of L. monocytogenes PI-PLC needed in
the absence of the BR-PLC.
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FIG. 3.
Formation of [3H]IP3 in J774
cells prelabeled with [3H]inositol after infection with
L. monocytogenes strains. [3H]IP3
was extracted from infected cells at 30 min after infection and
separated by ion-exchange chromatography as described in the text. The
data represent the means ± the SEM for wild-type (n = 6), plcA (n = 5), and hly (n = 5)
strains and, in a separate set of experiments, for wild-type
(n = 3), plcB (n = 3), and plcA
plcB (n = 3) strains. The difference between
wild-type and hly strains was significant (P < 0.01, Mann-Whitney).
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Activation of host PLC also occurs in murine bone marrow-derived
macrophages infected with L. monocytogenes.
The formation of
[3H]IP was also observed in murine bone marrow-derived
macrophages infected with the wild-type strain with a time course
similar to that observed with J774 cells. As in J774 cells, the
formation of [3H]IP was much lower with the PI-PLC mutant
(Fig. 4A). The activation of host
phospholipases by the wild type was also evidenced by the formation of
IP3. Infection with the PI-PLC mutant resulted in as much
IP3 formation as did infection with the wild type (Fig. 4B). Earlier experiments in which bone marrow-derived macrophages were
infected with L. monocytogenes grown to the stationary phase showed that a mutant in LLO produced similarly reduced levels of
[3H]IP as the PI-PLC mutant, both of which were much
lower than was observed with the wild type (data not shown).
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FIG. 4.
Formation of [3H]IP and
[3H]IP3 in murine bone marrow-derived
macrophages prelabeled with [3H]inositol after infection
with L. monocytogenes. Radioactive products of PI hydrolysis
(IP) (A) and PIP2 hydrolysis (IP3) (B) were
extracted from infected cells at the times indicated and separated by
ion-exchange chromatography as described in the text. The data
represent the means ± the SEM of two separate experiments.
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Activation of host phospholipase D in J774 cells infected with
wild-type and mutant L. monocytogenes.
Many of the agonists
that stimulate eukaryotic polyphosphoinositide-specific PLC also result
in activation of PLD, and it appears that in some systems activation of
PLD occurs downstream of PLC activation. Furthermore, PLD has been
implicated in vesicular membrane traffic (31). Consequently,
we examined the activation of PLD in J774 cells infected with L. monocytogenes. Eukaryotic PLD catalyzes transphosphatidylation to
ethanol from various membrane lipids with phosphatidylcholine as the
preferred donor for the membrane-bound forms, which do not require
Ca2+ (31). Thus, in cells prelabeled with
[3H]palmitic acid, the formation of [3H]PEt
in the presence of ethanol serves as a measure of PLD activity. Infection with wild-type L. monocytogenes and all mutants
studied except for a mutant in LLO activated PLD to a similar extent in J774 cells (Fig. 5). These results were
obtained at 90 min after infection. The kinetics of activation of PLD
upon wild-type infection of J774 cells and upon infection with all
mutants except the LLO deletion strain were similar, and the results of
a typical experiment are shown in Fig. 6.
Several experiments showed that there was no detectable increase in PEt
during the first 15 min of infection (data not shown); however, PLD
activity at 30 min was readily seen.
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FIG. 5.
Formation of [3H]PEt in J774 cells
prelabeled with [9,10-3H]palmitate at 90 min after
infection with L. monocytogenes strains.
[3H]PEt was extracted from the cells and separated by
thin-layer chromatography as described in the text. The data represent
the means ± the SD of two separate experiments carried out in
duplicate, in which each mutant strain was compared with the wild type
in the same experiment. For wild type, n = 22; for all
other strains, n = 4. The difference between the
wild-type and the hly infections was highly significant
(P < 0.0001, unpaired t test). The
difference in [3H]PEt between uninfected control cells
and the cells infected with the hly strain was not
significant.
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FIG. 6.
Formation of [3H]PEt in J774 cells
infected with wild-type L. monocytogenes and with the
hly strain. The data are from one of two experiments with
the hly strain, each done in duplicate.
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Evidence for the participation of PKC in the activation of PLD has been
obtained in several experimental systems; however,
the results have
been mixed (
10,
31). As in many cell systems,
short-term
activation of PKC by phorbol 12-myristate 13-acetate
(PMA) resulted in
a time-dependent activation of PLD (Table
1).
In agreement with this, overnight
pretreatment with PMA decreased
the activation of PLD upon wild-type
infection to a nearly basal
level (Table
2). Also in agreement, the treatment of
cells with
rottlerin (Calbiochem), an inhibitor of PKC
, resulted in
strong
inhibition of the PLD activation caused by
L. monocytogenes infection.
However, hispidin (Calbiochem), which
inhibits both PKC
I and
II, but not PKC
, had no effect on PLD
activation (Table
3).
Both PKC inhibitors
partially inhibited escape from the primary
phagocytic vacuole
(Wadsworth and Goldfine, unpublished).
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TABLE 2.
Overnight (18 h) pretreatment with PMA inhibits
activation of PLD with wild-type L. monocytogenes in
J774 cells
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Inhibition of PLD by the competitive inhibitor 2,3-DPG results in
decreased escape from the primary vacuole.
We studied the effects
of the competitive PLD inhibitor 2,3-diphosphoglycerate (2,3-DPG)
(20) on PLD activation in J774 cells upon infection with
wild-type L. monocytogenes. Treatment of uninfected J774
cells with 2,3-DPG surprisingly resulted in variable stimulation of
basal PLD activity (5 mM, 151% ± 59% of the no-drug control; 10 mM, 212% ± 183% of the control). After infection, PLD
activation was inhibited 91% ± 19% by 5 mM 2,3-DPG and
86% ± 12% by 10 mM 2,3-DPG (three separate experiments at each
concentration, carried out in duplicate). Measurement of the effects of
2,3-DPG treatment on the escape from the primary vacuole of wild-type
bacteria showed significant inhibition at both concentrations. The PLD
inhibitor 2,3-DPG was found to partially inhibit escape from the
primary vacuole as follows: with no pretreatment, there was 45% escape
in 1.5 h; with 2,3-DPG at 5 and 10 mM, the percent escape values
were 29 and 23%, respectively. (The percent escape values refer to the
percentage of internalized bacteria associated with polymerized actin
at 1.5 h. The data were pooled from two separate experiments.)
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DISCUSSION |
The observations that infection of murine macrophage-derived J774
cells with wild-type L. monocytogenes resulted in two brief elevations of intracellular Ca2+ within the first 5 min of
infection, followed by a third prolonged elevation lasting for more
than 30 min, and that the same pattern of calcium fluxes was not
observed upon infection with mutants in LLO, PI-PLC, or BR-PLC led to
the questions concerning the origins of these fluxes and their effects
on other host cellular activities. The importance of these observations
was heightened by the finding that pharmacological inhibition of the
induced calcium signals resulted in changes in the kinetics of
bacterial uptake into these cells and the efficiency of escape from the primary phagocytic vacuole (36). In parallel with studies on calcium signaling in J774 cells, we have carried out studies on the
hydrolysis of host phosphoinositides and the activation of PLD induced
by wild-type and mutant L. monocytogenes.
The observed formation of [3H]IP in cells prelabeled
overnight with [3H]inositol beginning during the first 10 min of infection was, like the first and second elevations of
intracellular calcium ([Ca2+]i), dependent on
the expression of both LLO and PI-PLC. Early formation of
[3H]IP did not require the expression of BR-PLC, which is
not required for the first, brief elevation of
[Ca2+]i (36). Therefore, the
observed increase in [3H]IP release is likely to result
from the activity of bacterial PI-PLC. It does not appear to result
from activation of host PLCs, which preferentially hydrolyze PI-4-P and
PI-4,5-P2 to produce IP2 and IP3,
and subsequent hydrolysis of these by phosphatases to yield IP. This
conclusion is based on our finding that infection with the double
plcA plcB mutant lacking both PI-PLC and BR-PLC caused
accumulation of IP2 and IP3 equal to that of
wild-type infection in J774 cells but did not result in increased IP
formation (Fig. 2 and 3).
We had previously hypothesized that the first brief
[Ca2+]i elevation resulted from activation of
the calcium-independent PKC found in J774 cells (32)
upon release of DAG formed by the action of PI-PLC. Presumably, either
pores or membrane perturbations induced by LLO permit access of PI-PLC
to host PI (30, 36). Our measurements of PI hydrolysis show
release of [3H]IP during the first 10 min of infection,
but the background level in J774 cells is too high to measure early
release of either IP or DAG (33). However, studies on PKC
show that it is activated within the first min of infection of J774
cells with wild-type L. monocytogenes and that its
activation required expression of both LLO and PI-PLC (Wadsworth and
Goldfine, unpublished). Thus, there is excellent agreement between
rapid PKC activation, the earliest inward Ca2+ flux,
and the hydrolysis of host PI, with the ability of L. monocytogenes to express LLO and PI-PLC.
The more rapid release of [3H]IP beginning between 20 and
30 min after infection mirrors the kinetics of internalization of the
wild type, which is inhibited during the first 5 min and approaches a
plateau at 20 min after infection. It also corresponds temporally with
the third elevation of [Ca2+]i
(36). Enhanced activity of bacterial PI-PLC after entry into the phagosome is presumably the result of the increased expression of
LLO and PI-PLC observed after bacterial internalization (6, 19,
22). This increased activity was presumably not dependent on
elevated [Ca2+]i since it was observed with
the BR-PLC deletion mutant, which produces only a brief spike in
[Ca2+]i at the first minute after infection.
This is consistent with the lack of a Ca2+ requirement for
bacterial PI-PLCs in general and for that of L. monocytogenes (16, 23).
Activation of host PLC.
The release of IP2 and
IP3, indicating activation of host phosphoinositide PLC,
was also dependent on expression of LLO. In most experiments with J774
cells and the PI-PLC deletion mutant, the release of IP2
and IP3 was lower than wild-type, but there was
considerable scatter in these data. Indeed, PI-PLC expression was not
required for host PLC activation in the absence of BR-PLC (Fig. 3),
which suggests that a product of BR-PLC is inhibitory for host PLC
activation. These could include DAG derived from lipids other than PI
(37), ceramide, or metabolic products derived from these
lipid intermediates. Since the mutant lacking both PI-PLC and BR-PLC
did not produce any [Ca2+]i elevation in J774
cells (36), elevated [Ca2+]i is
not a requirement for activation of host PLC in this system. Eukaryotic
PLCs require Ca2+ for catalytic activity and are activated
by elevated [Ca2+]i, but activation by the G
protein-dependent pathway occurs in the resting physiological range of
~100 nM (2, 31), which is the level we have observed in
uninfected J774 cells (36). It is also important to note
that the presence of bacterial PI-PLC, which causes hydrolysis of PI
and could thus affect the pool of cellular PI-4,5-P2, does
not interfere with the stimulation of IP3 release.
Surprisingly, there does not appear to be a connection between the
activation of host PLC, which gives rise to IP
3, a mediator
of release of Ca
2+ from intracellular stores, and the
elevations of [Ca
2+]
i observed in J774 cells
after infection. Infection with the
double phospholipase mutant of
L. monocytogenes resulted in the
same level of host PLC
activation as with the wild type, yet it
produced no elevation of
[Ca
2+]
i (
36). Release of calcium
from intracellular stores in these
cells may result from signaling
molecules other than IP
3. One
candidate is sphingosine-1-P
(
15,
34), which can be produced
as a result of the formation
of ceramide by the action of
L. monocytogenes BR-PLC
(
33), followed by hydrolysis of the
N-acyl group
and
phosphorylation of the resulting sphingosine. Some evidence for
this hypothesis is the finding that an
L. monocytogenes
construct
in which the gene for the PC-PLC of
B. cereus,
which has essentially
no sphingomyelinase activity, replaced
plcB (
39) produced much
lower
[Ca
2+]
i levels than did the wild type after
10 min of infection of
J774 cells (Wadsworth and Goldfine,
unpublished).
Activation of host PLD.
Of the mutants studied, only the
strain deficient in LLO was unable to activate host PLD. Thus,
bacterial PI-PLC and BR-PLC activities and elevated host
[Ca2+]i are not required for PLD activation,
since the double phospholipase mutant activated PLD but did not
increase [Ca2+]i (36). Short-term
treatment with PMA stimulated PLD to the same level observed on
infection with L. monocytogenes and overnight pretreatment
with PMA inhibited PLD activation (Table 1), suggesting an involvement
of PKCs in the activation of PLD. The finding that rottlerin, an
inhibitor of PKC in J774 cells, strongly inhibited PLD activation
is also consistent with a role for PKC in PLD activation; however,
hispidin, an inhibitor of both PKC I and II, but not PKC , did
not block PLD activation in infected J774 cells (Table 3). These
findings are consistent with many observations suggesting a connection
between PKCs and mammalian PLC and PLD activation; however, there is no
established mechanism for activation of PLD by PKC, and the results
with inhibitors have been variable (31). The finding that
PLD activation occurred after 20 min of infection compared to rapid
changes in [Ca2+]i and rapid activation of
PKC isoforms (Wadsworth and Goldfine, unpublished) suggests that PLD
activation is downstream of these signals and may involve more
complicated and possibly parallel pathways (2, 8).
The observation that PLD activation, like escape from the primary
vacuole, is dependent on LLO expression and occurs during
the time when
the phagosome is beginning to undergo lysis suggests
a connection
between these phenomena. This notion is bolstered
by the finding that
the PLD inhibitor 2,3-DPG resulted in decreased
escape from the primary
vacuole. However, PI-PLC loss results
in decreased efficiency of escape
from the primary vacuole (
7,
33,
36) but does not affect PLD
activation. Also, as noted
above, hispidin partially inhibits escape
from the primary vacuole
but had no effect on PLD activation. Kusner et
al. have observed
a tight coupling of phagocytosis of
M. tuberculosis and opsonized
zymosan and activation of PLD in human
macrophages (
20). At
this time it is not possible to state
that similar tight coupling
exists in the fate of
L. monocytogenes during infections. Clearly,
further studies will be
required to dissect these complex and
long-range
interactions.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the National Institutes of
Health (GM52797) and the University Research Foundation of the
University of Pennsylvania.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, School of Medicine, University of Pennsylvania, 301C
Johnson Pavilion, Philadelphia, PA 19104-6076. Phone: (215) 898-6384. Fax: (215) 573-4856. E-mail:
goldfinh{at}mail.med.upenn.edu.
Editor:
E. I. Tuomanen
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Abstract
Introduction
