Full text of DOI:10.1016/S1286-4579(02)00011-4

Microbes and Infection 4 (2002) 1335–1343
www.elsevier.com/locate/micinf
Review
Macrophage intracellular signaling induced by Listeria monocytogenes
Howard Goldfine *, Sandra J. Wadsworth 1
Department of Microbiology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, PA 19104-6076, USA
Abstract
Macrophages are critical for control of Listeria monocytogenes infections; accordingly, the interactions of L. monocytogenes with these
cells have been intensively studied. It has become apparent that this facultative intracellular pathogen interacts with macrophages both prior to
entry and during the intracellular phase. This review covers recent work on signaling induced in macrophages by L. monocytogenes, especially
intracellular signals induced by secreted proteins including listeriolysin O and two distinct phospholipases C.
© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
Keywords:Listeria monocytogenes; Intracellular signaling; Macrophage; Phospholipase C; Protein kinase C
1. Introduction
The doubling time of Lm in the cytoplasm of macrophage-
like cells in vitro is about 1 h; however, in the liver, it is 3–4 h
[3].
Listeria monocytogenes (Lm), a food-borne pathogen of
humans and other animal species, encounters a variety of cell
types after being consumed in contaminated food, first in its
passage through the digestive tract, then the liver, the circu-
latory system, and the central nervous system, often with
fatal consequences [1,2].A murine model of infection, which
introduces Lm into the bloodstream, results in rapid infection
of the liver and spleen. In the liver most of the bacteria are
killed upon initial encounter with Kupffer cells, resident
macrophages in the liver, or with immigrant macrophages.
Since some Lm can survive this encounter, neutrophils play
an important role in resolving these infections [3,4]. Depend-
ing on the experience of the host, the proportion of surviving
bacteria will vary. In studies with isolated macrophages and
macrophage-derived cell-lines, the surviving bacteria escape
from the primary phagocytic vacuole, grow and divide in the
cytoplasm, recruit and polymerize host cell actin, which
provides the driving force for movement through the cyto-
plasm and into nearby cells by means of filopodia-like pro-
jections [5]. In these neighboring cells bacteria escape from
double-membrane secondary phagosomes and repeat the
cycle of growth, recruitment of actin and cell-to-cell spread.
The secretion of cytokines by resident and migrant cells
during the early stages of Lm infections represents an impor-
tant means of controlling infection through macrophage acti-
vation, the recruitment of neutrophils, natural killer cells and
T cells. Wagner and Czuprynski observed temporally distinct
groups of cytokine gene expression in the livers of infected
mice. IFN-c, TNF-a and IL-10 were induced within 1 day
after challenge. IL-2 and IL-4 were strongly expressed at 1
day but suppressed by 3 days after infection. Other cytokines
were expressed later [6].
Lm secretes listeriolysin O (LLO) and two distinct phos-
pholipases C (PLC), one specific for phosphatidylinositol
(PI) and glycosyl-PI (GPI)-anchored proteins (PI-PLC)
[7–9], and a second broad-range phospholipase (BR-PLC)
capable of hydrolyzing a wide variety of mammalian phos-
pholipids, including sphingomyelin [10,11]. Based on his-
torical precedents, this type of PLC is often designated
phosphatidylcholine-specific or -preferring PLC and abbre-
viated PC-PLC, but we will use BR-PLC throughout, since
there is no strong preference for PC [12]. These three pro-
teins are encoded by genes in a compact regulon on the
chromosome of Lm, and their expression is controlled by
PrfA, a 27 kDa protein, which binds to their promoters and
positively regulates their expression (for a recent review see
[13]). A number of studies have provided evidence for the
participation of both PLCs in addition to LLO in the induc-
tion of host intracellular signaling. In this review we briefly
* Corresponding author. Tel.: +1-215-898-6384; fax: +1-215-898-9557.
E-mail address: goldfinh@mail.med.upenn.edu (H. Goldfine)
1
Present address: Department of Pharmacology, Temple University
School of Medicine, Philadelphia, PA 19140-5104, USA.
© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
PII: S 1 2 8 6 - 4 5 7 9 ( 0 2 ) 0 0 0 1 1 - 4

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H. Goldfine, S.J. Wadsworth / Microbes and Infection 4 (2002)
describe the properties of these proteins and their roles in
various interactions with mammalian macrophages and with
other important host cells. Host cell signaling induced by
internalins has been reviewed recently [14,15], and will not
be discussed.
were less efficient than the wild type in escape from the
primary phagocytic vacuoles of murine bone marrow-
derived macrophages [28,29] and the J774 murine
macrophage-like cell line [30]. They also show a small defect
in cell-to-cell spread [28,31]. The deletion mutant displayed
a significant growth defect in the murine liver but not the
spleen [28]. The orthologous enzymes from Gram-positive
bacteria are similar in size to that of Lm. By amino acid
sequence, the two enzymes from Bacillus cereus (Bc) and B.
thuringiensis (Bt)are closely related to each other. The PI-
PLC from Staphylococcus aureus is more closely related to
those from Bc and Bt than the Lm enzyme [32,33]. Among
Listeria species, homologous genes have only been found in
L. ivanovii, an animal pathogen, and in L. seeligeri, but not in
other non-pathogenic species [4]. The purified Lm PI-PLC is
highly active on PI, but, unlike the enzymes from Bacillus
and that from S. aureus [32], its ability to cleave GPI-
anchored proteins is relatively limited, with specific activi-
ties from 0 to 25% of that of the enzyme from Bt, depending
on the GPI-anchored protein [34]. The crystal structure of Bc
PI-PLC in complex with glucosaminyl (a1#6)D-myo-
inositol, the structure common to all GPI-anchors, showed
that the glycan portion of GPI interacts with a shallow groove
extending from the active site [35].A boundary of this groove
is completely missing from Lm PI-PLC [33], resulting in a
large widening of the binding site which probably causes a
significant reduction of interactions with the glycans of GPI-
anchored proteins [33]. Aside from this difference, and de-
spite relatively low amino acid identities (~24%), the two
structures are highly homologous, except in several loop
regions that accommodate most of the amino acid insertions
and deletions. The active sites are also well conserved [33],
and mutagenesis of two highly conserved histidines in the
active site of Lm PI-PLC resulted in loss of both catalytic
activity of the enzyme and its biological activity in tissue
culture models of infection [29]. Since PI-PLC from Bc
binds to GPI-anchors 50–100 times more strongly than to PI
[36], the opening of the binding site in the Lm protein may
have evolved to permit fewer interactions with these sub-
strates and a greater focus of activity on PI, which could have
significant consequences, as will be discussed below.
2. Listeria monocytogenes proteins involved in host
signaling
2.1. Listeriolysin O (LLO)
LLO, 58 kDa, encoded by hly, is a member of a large
family of oxygen-labile, sulfhydryl-activated cytolysins
present in many Gram-positive bacteria, among the best
studied of which are streptolysin O (SLO), perfringolysin O
(PFO), and LLO [16]. These proteins insert into eukaryotic
cell membranes, apparently upon binding to cholesterol.
Monomers polymerize to form pores of varying sizes de-
pending on the available concentration of monomer. The size
of the average pore formed by SLO, 10–20 nm, is sufficient
to allow leakage of macromolecules from affected cells, and
similar pores have been seen with LLO [17]. Evidence for the
ability of SLO to serve as a conduit for proteins secreted by
Streptococcus pyogenes into host cells has recently been
presented [18]. Lm mutants lacking LLO are unable to es-
cape from the primary phagocytic vacuole, and, conse-
quently fail to grow in infected cells. These mutants are
essentially avirulent in mice (reviewed in [3,4]). Unlike other
members of this family of cytolysins, which are most active
at neutral pH, LLO has a pH optimum at 5.5 [19]. This
property is consistent with its role in the acidified phago-
some; however, as will become apparent in this review, its
activity at neutral pH is not negligible, and many of its
activities occur under the neutral pH conditions expected to
be found in the extracellular space.
During invasion of host cells, the synthesis of LLO is
increased in the phagosome [20,21] and it is further increased
when the bacteria escape into the cytosol, but there it is
rapidly degraded [20]. Recent work has implicated a PEST-
like sequence in promoting the degradation of this potently
cytotoxic molecule [22,23]. Experiments with LLO mutants
coated with LLO have shown that it is also required for lysis
of the secondary vacuoles formed upon cell-to-cell spread
[24]. The crystal structure of LLO has not been solved, but
that for the related PFO has [25], and models for insertion of
PFO into eukaryotic membranes have been summarized [26].
2.3. Broad-range PLC
BR-PLC of Lm, encoded by plcB, is related to similar
Zn2+-dependent enzymes secreted by Gram-positive bacteria
such as B. cereus, Clostridium perfringens (a-toxin), and
Clostridium bifermentans[10,11,37]. The Lm enzyme,
29 kDa, is similar in size to that from Bc and shares 39%
amino acid identity [11]. An insertion mutation in plcB re-
sulted in a significant increase in bacteria in the double-
membrane vacuoles resulting from cell-to-cell spread, and
this mutant and a deletion mutant produced plaques of re-
duced size on fibroblast monolayers. However, escape from
the primary phagocytic vacuole of macrophages was normal
[11,31]. Thus, it appears that the BR-PLC plays an important
2.2. Phosphatidylinositol-specific PLC
This highly specific phospholipase C, 33 kDa, encoded by
plcA, is a member of a family of bacterial PLCs that cleave
PI. Eukaryotic enzymes specific for phosphoinositides pref-
erentially hydrolyze phosphorylated forms of PI, e.g. PI-4-P
and PI-4, 5-P2[27], whereas the bacterial PI-PLCs are not
active on phosphorylated forms of PI. Lm mutants in PI-PLC

H. Goldfine, S.J. Wadsworth / Microbes and Infection 4 (2002)
1337
Table 1
Patterns of calcium elevations and PKC translocation observed in J774 cells upon infection with L. monocytogenes wild type and mutants. Effects of
pharmacological agents
Strain
1st Ca2+ elevation, 2nd Ca2+ elevation, 3rd Ca2+ elevation, PKC d Translocation, PKC b II Transloca-
PKC b I Translocation,
1 to 4 min p.i.
1sta min p.i.
5 min p.i.
10 min p.i.
1sta min p.i.
tion, 1sta min p.i.
Wild type
+
–
–
+
–
–
+
–
–
–
–
–
+
+
–
–
+
–
+
+
–
–
+
–
–
+
–
LLO–
–
PI-PLC–
BR-PLC–
PI-PLC–, BR-PLC–
Wild type, SK&F
96 365b, c
Wild type, rottlerinc
Wild type, Hispidinc
+/–d
–
+
+
+
+
–
–
–
N.D.e
N.D.e
N.D.e
N.D.e
–
–
–
+
+
+
–
a Post infection.
b Calcium channel blocker.
c Cells were pretreated with pharmacological agents prior to and during infection.
d Indicates a relatively low elevation of intracellular calcium relative to that induced by the wild type.
e Not done.
role in escape from the secondary vacuole. A double plcA,
plcB deletion mutant produced much smaller plaques than
either of the single mutants, and the result was more than
additive. These results led to the prediction that PI-PLC also
plays a significant role in escape from the secondary vacuole
[31]. BR-PLC participates in escape from the primary vacu-
ole in non-phagocytic cells such as the Henle 407 epithelial
cell line, and in the absence of LLO, BR-PLC is capable of
mediating escape in these cells [38,39]. The overlapping
functions of the two phospholipases was also apparent in the
murine model of infection. When Lm was delivered i.v., a
plcA deletion had a barely measurable effect on virulence in
the mouse, a plcB deletion resulted in a 10–20-fold increase
in LD50, whereas the double mutant displayed a 250–500-
fold increase in LD50[31].
ciency for escape from the primary vacuole of Henle 407
epithelial cells in a strain lacking LLO, suggesting that leci-
thinase rather than sphingomyelinase is more important for
this function [40].
3. Intracellular signaling induced by L. monocytogenes
3.1. Calcium signaling
Three elevations of intracellular calcium ([Ca2+]i) occur
immediately after infection of J774 cells with wild-type Lm.
The first begins within 1 min after addition of a washed
suspension of bacteria in PBS to a monolayer of J774 cells in
the presence or absence of serum.After a brief decline to near
basal levels, [Ca2+]i increases again at 5–7 min post-infection
(p.i.), declines and then increases for a prolonged period
starting at 15 min p.i. Infection with a LLO mutant produces
none of these calcium fluxes. A PI-PLC mutant produced a
relatively weak signal beginning at 15 min that lasted for
about 15 min, and a BR-PLC mutant produced the first spike
in [Ca2+]i, but not the others (Table 1) . The first and second
elevations of [Ca2+]i occur at a time when very few wild-type
bacteria have been internalized, and these calcium spikes are
seen in cells containing no bound or internalized bacteria
[30]. Furthermore, similar signals, although somewhat de-
layed, could be obtained when bacteria were separated from
J774 cells by a membrane that permitted proteins, but not
bacteria, to penetrate (Wadsworth, unpublished). Thus, it
appears that calcium signaling can be induced by secreted
factors.
Activation of BR-PLC requires processing by a protease
to cleave the inactive proenzyme. In culture supernatants this
function is assumed by Mpl, the product of the mpl gene;
however, in infected cells a putative host protease can fulfill
this function [39]. Lm BR-PLC differs from Bc PC-PLC in
that the former is capable of hydrolyzing sphingomyelin in
addition to phosphoglycerolipids [10]. Hydrolysis of PC and
phosphatidylethanolamine occurs at comparable rates, but
hydrolysis of sphingomyelin is somewhat slower [12]. The
activity of Bc PC-PLC on its preferred substrate, PC, is
approximately 10-fold higher than that of Lm BR-PLC. The
Bc enzyme is strongly inhibited by halides, especially the
larger atoms like iodine. Activity is also strongly diminished
by chloride at the concentration found in normal saline. This
is not the case for the Lm enzyme, which was moderately
stimulated by halide salts [12]. A Lm strain in which plcB
was replaced by the gene for PC-PLC from Bc had slightly
reduced virulence in mice compared to wild-type Lm, but it
was more virulent than the strain containing a plcB deletion
and produced plaques of intermediate size, a measure of the
ability to spread from cell-to-cell. Thus the ability to cleave
sphingomyelin appears to be significant for Lm virulence.
The strain containing Bc PC-PLC displayed increased effi-
All three [Ca2+]i elevations were prevented by pretreat-
ment of host cells with the receptor-operated calcium chan-
nel blocker SK&F96365 or by removal of extracellular cal-
cium by chelation with EGTA. Pretreatment with
thapsigargin, an inhibitor of calcium release from intracellu-
lar stores, prevented only the second and third elevations.
Together, these results indicated that the first calcium eleva-

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H. Goldfine, S.J. Wadsworth / Microbes and Infection 4 (2002)
tion resulted from influx of extracellular calcium through a
channel and that the latter two elevations reflected release of
calcium from intracellular stores, which required the initial
calcium influx.
and IP3, resulting from activation of host PLC isoforms [44].
DAG was also observed to accumulate rapidly. Since a simi-
lar response was elicited by LLO alone, it appears that host
PLC isoforms were activated [45]. A model was presented in
which pores formed by LLO permit access of PI-PLC to PI in
the inner leaflet of the host cell plasma membrane, resulting
in the formation of IP, and a separate induction of host
phospholipases [44]. From studies with J774 cells, it appears
likely that LLO induces a potent phosphoinositide response,
in the absence of sustained calcium elevation, since the
double PI-PLC, BR-PLC deletion mutant of Lm, which pro-
duced no intracellular Ca2+ elevation [30], evoked release of
IP2 and IP3, but an LLO– strain did not [41].
In both HUVEC [44,45] and human neutrophils [46] Lm
and L. innocua strains expressing LLO induced the release of
platelet activating factor and products of arachidonate me-
tabolism, prostaglandin I2 or leukotrienes. Indicating activa-
tion of host phospholipase A2. In HUVEC, a combination of
LLO and PI-PLC were required for maximal responses, but
in neutrophils, the response was mainly attributed to LLO.
The entry of wild-type Lm into J774 cells is slower than
the entry of the LLO and PI-PLC mutants. Like heat-killed
bacteria, these mutants are taken up rapidly so that from 50 to
70% of bacteria associated with the host cells are internalized
within 1 min after infection compared to 10–15% of the wild
type. Preventing the elevation of [Ca2+]i with either
SK&F96365 or thapsigargin increased the rate of entry of the
wild type, but not to that observed with the LLO and PI-PLC
mutants. Treatment with either inhibitor also resulted in
greatly decreased efficiency of escape of wild-type Lm from
the primary phagocytic vacuole [30]. As will become appar-
ent below, calcium signaling is linked to protein kinase C
(PKC) mobilization, which also modulates uptake and es-
cape from the phagosome.
3.2. Induction of host phospholipases A and C
3.3. Mobilization of host PKC isoforms
The ability of wild-type Lm to induce calcium fluxes in
J774 macrophage-like cells and the finding that the second
brief elevation and the third prolonged elevation were inhib-
ited by thapsigargin, which depletes calcium stores, indi-
cated that these calcium fluxes resulted from release of Ca2+
from intracellular stores [30]. An important mediator of Ca2+
release from the endoplasmic reticulum is inositol-1, 4, 5-P3
(IP3), a product of hydrolysis of PI-4, 5-P2 (PIP2) by eukary-
otic calcium-activated PLC. Studies with murine bone
marrow-derived macrophages have shown distinct elevation
of IP2 and IP3 on infection with wild-type Lm, but not with an
LLO– mutant. Infection of J774 cells prelabeled with [3H]-
inositol with wild-type Lm resulted in elevation of [3H]-IP
within 10 min, followed by more rapid release at 20–30 min.
Much less [3H]-IP formation was observed upon infection
with mutants deficient in either LLO or PI-PLC, but loss of
BR-PLC did not affect IP formation. It appears that PI-PLC
with the aid of LLO rapidly hydrolyzes host PI, resulting in
release of diacylglycerol (DAG) and IP [41].
Release of IP3, unlike release of IP, is an indicator of host
PLC activation, since the Lm PI-PLC does not hydrolyze
PIP2[42,43]. IP3 elevation occurred within 10 min of infec-
tion of J774 cells—with wild-type Lm, but not with an LLO
mutant. IP3 elevation was somewhat reduced with a PI-PLC
mutant. However, a mutant deficient in BR-PLC and a double
PI-PLC, BR-PLC mutant activated host PLC fully, suggest-
ing that BR-PLC or product(s) of its activity directly or
indirectly inhibit host PLC activation [41].
Since hydrolysis of PI by bacterial PI-PLC leads to the
production of DAG, and both DAG and calcium are known
activators of PKC, activation of PKC isoforms was postu-
lated to occur in infected host cells [30]. The four major
isoforms of PKC found in the J774 murine macrophage-like
cells are a, bI, bII, and d [47]. Of these, a, bI, and bII are
“classical” isoforms activated by intracellular calcium and/or
DAG, while PKC d is a “novel” isoform which is activated by
DAG, but is Ca2+-independent [48]. PKC d has been impli-
cated in phosphorylation and opening of Ca2+ channels in
eukaryotic cells [49,50].
Using immunofluorescence, we observed that PKC d
moves to a peripheral location within 30 s of addition of a
washed suspension of wild-type Lm or the BR-PLC mutant
to J774 cells, but not after addition of either LLO or PI-PLC
deletion mutants. This translocation is calcium-independent,
since it was not inhibited by SK&F96365. It was inhibited by
3 h pretreatment with phorbol myristate acetate (PMA),
which downregulates PKCs. Pretreatment with PMA or rot-
tlerin, an inhibitor of PKC d, abolished the first calcium
signal, consistent with the role of PKC d in activating cal-
cium channels [51].
Immunofluorescence studies have revealed rapid translo-
cation of PKC bI and bII to early endosomes after infection
of J774 cells with wild-type Lm or the BR-PLC mutant. The
translocation of PKC bII was seen from 30 s to 3 min p.i.,
while that of PKC bI occurred between 1 and 4 min p.i. No
PKC bI or bII translocation was observed after infection with
a LLO mutant. PKC bI, but not PKC bII, translocation to
early endosomes occurred after infection with a PI-PLC
mutant. Isolation of early endosomes by centrifugation of a
mixed membrane fraction on Percoll gradients followed by
separation of early endosome proteins by gel electrophoresis
Earlier studies with human umbilical vein endothelial
cells (HUVEC), which had not internalized Lm during the
course of these experiments, showed that a combination of
LLO and Lm PI-PLC secreted by recombinant Listeria in-
nocua strains, resulted in the formation of products of phos-
phoinositide hydrolysis designated IPx, which were presum-
ably a mixture of IP, resulting from hydrolysis of PI, and IP2

H. Goldfine, S.J. Wadsworth / Microbes and Infection 4 (2002)
1339
Fig. 1. A proposed PKC signaling pathway in J774 cells infected with L.monocytogenes. In this pathway diacylglycerol (DAG) generated by either bacterial
phosphatidylinositol specific phospholipase C (PI-PLC) or host PLC acting on PI-4, 5-P2 (PIP2) or other phosphorylated forms of PI, recruits PKC isoforms to
membranes, where they presumably become activated.(Note that the pores formed by LLO usually involve the assembly of many monomers, not the few shown
here for convenience). Adapted from [51], with permission from American Society for Microbiology.
and Western blotting with antibodies specific for PKC bI or
bII , produced results consistent with those obtained by
immunofluorescence. PKC translocation like the first cal-
cium elevation occurred in cells that had not internalized Lm
[51].
type, and drugs that block calcium signaling had minimal
effects on uptake of these mutants [30]. Hispidin, which
inhibits PKC b selectively, also increased the association of
wild-type Lm with J774 cells to the levels seen with the
PI-PLC mutant. The percent of wild-type bacteria internal-
ized was also increased, but at 1 min, it did not equal the
uptake of the PI-PLC mutant. It appears that PKC signaling
modulates uptake of Lm into these macrophage-like cells. As
was the case for inhibitors of calcium signaling [30], hispidin
also greatly decreased the efficiency of escape of wild-type
Lm from the primary vacuole. Hispidin treatment of the J774
cells infected with the PI-PLC mutant, which is less efficient
in escape from the primary vacuole than the wild type, almost
completely abolished escape at 1.5 h after infection [51].
Translocation of PKC bII to early endosomes is calcium-
and presumably DAG-dependent since, it was blocked by
treatment with the calcium channel blocker SK&F96365 and
required bacterial PI-PLC. It was also inhibited by hispidin, a
relatively specific PKC b inhibitor. Translocation of PKC b I,
in contrast, did not appear to require elevated intracellular
Ca2+, since it was seen upon infection with a double phos-
pholipase mutant (plcA, plcB), which produced no calcium
signal in J774 cells. DAG was presumably produced upon
infection with the double phospholipase mutant by activation
of host phosphoinositide-specific PLC [30,41].
3.4. Induction of host phospholipase D (PLD) activity
These results lead to a proposed signaling pathway in
which LLO and PI-PLC cooperate in cleavage of host PI,
resulting in translocation of PKC d to the cell membrane.
PKC d mobilization leads to Ca2+ influx, which in combina-
tion with DAG, results in translocation of PKC bII to early
endosomes (Fig. 1 ). Lm mutants that do not secrete phos-
pholipases, but do secrete LLO, can activate PKC bI through
activation of host phospholipases, which also produce DAG.
Activation of eukaryotic PLD is often obtained with ago-
nists that activate PLC isoforms, and in some systems activa-
tion of PLD occurs downstream of PLC [52,53]. Infection of
J774 cells with wild-type Lm and with mutants lacking either
or both of the Lm PLCs resulted in activation of host PLD. Of
the strains tested, only that lacking LLO failed to activate
PLD. This increase in PLD activity occurred 15 min p.i. and,
therefore, after translocation of PKC bI, bII, and d. It is either
coincident with or following activation of host
polyphosphoinositide-specific PLC [41].
As noted above, inhibition of calcium signaling with ei-
ther SK&F96365 or thapsigargin resulted in increased early
association of wild-type Lm with J774 cells and increased
uptake of associated bacteria. PI-PLC or LLO mutants asso-
ciate with J774 cells and enter more rapidly than the wild
PLD activity was also stimulated in J774 cells by short-
term treatment with PMA, an activator of PKC, and its

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H. Goldfine, S.J. Wadsworth / Microbes and Infection 4 (2002)
stimulation by Lm infection was nearly abolished by long-
term treatment with PMA, which results in loss of PKC,
suggesting a role for PKC in PLD activation in this system.
However, hispidin, which inhibits PKC b I and II activation
in these cells did not inhibit PLD activation [41].
activation occurred prior to entry of the bacteria [60]. Infec-
tion of HUVEC results in translocation of NF-jB to the
nucleus and upregulation of adhesion molecules such as
E-selectin, VCAM-1 and ICAM-1 [61–63]. There is general
agreement that LLO is required for full NF-jB activation in
HUVEC. The need for bacterial phospholipase expression,
which was correlated with the ability to produce ceramide,
was reported in one of these studies [62], but not in the other
study that examined this requirement [63]. The phospholi-
pase effects could be reproduced by addition of either cera-
mide or sphingomyelinase [62].
The activation of PLD in J774 cells coincides with the
entry of wild-type Lm into host cells and the beginning of
vacuolar maturation [30]. Inhibition of PLD by 2,3-
diphosphoglycerate was accompanied by decreased effi-
ciency of escape from the primary vacuole [41]. Since the
loss of PI-PLC by mutation also results in diminished effi-
ciency of escape, but does not affect PLD activation, PI-PLC
probably contributes to the escape process through another
pathway, possibly that involving PKC activation. There is a
tight coupling between phagocytosis of Mycobacterium tu-
berculosis as well as opsonized zymosan particles and PLD
activation in human macrophages [54]. PLD activation and
the product of its activity, phosphatidic acid, are thought to
play important roles in intracellular vesicle transport, but the
mechanisms have not been established [53,55].
4. Signaling from outside and from within
the macrophage
From studies with macrophages, HUVEC and neutrophils
it has become apparent that proteins secreted by Lm includ-
ing LLO, PI-PLC and BR-PLC are capable of eliciting a
variety of cellular responses from outside the host cell. Some
of these responses are seen within the first minute of addition
of Lm to primary cells or cell lines and can take place prior to
uptake [30,46], when there is no uptake [44,45], or when
uptake is inhibited. For example, when the uptake of Lm into
neutrophils was blocked by treatment with botulinum C2
toxin, elastase secretion was stimulated [46], and when up-
take into J774 cells was suppressed by treatment with cy-
tochalasin D, early calcium signaling was still observed
(Wadsworth, unpublished).
The PKC signaling pathway diagramed in Fig. 1 results
from calcium-independent activation of PKC d, presumably
by DAG, which in turn is presumed to be responsible for the
initial elevation of [Ca2+]i seen within the first minute of
addition of Lm to J774 cells. Elevated [Ca2+]i combined with
DAG then promotes the translocation of PKC bII to early
endosomes. Blockage of this pathway, either by preventing
the elevation of [Ca2+]i or by inhibiting PKC b mobilization,
results in greater association of Lm with J774 cells and much
more rapid internalization of associated bacteria. The con-
nections between PKC activation and binding and uptake of
Lm are not understood.
Once Lm is internalized in host cells, the expression of
both LLO and PI-PLC increases [20,21,64]. In macrophages
transcription of hly and plcA was observed predominantly
within the phagosomal compartment [21], and the activity of
LLO is expected to increase as the phagocytic vacuole be-
comes acidified. Uptake into the phagocytic vacuole is con-
comitant with an increased rate of hydrolysis of PI and
activation of host PLD [41], suggesting that signaling contin-
ues between the time of entry and escape from the vacuole.
Blockage of the PKC signaling pathway either by inhibiting
calcium elevation [30] or by inhibiting PKC b mobilization
with hispidin results in decreased escape from the phago-
some at 1.5 h after infection [51]. If hispidin is removed after
entry, escape is normal, indicating the importance of contin-
ued PKC signaling after Lm is internalized (Wadsworth,
3.5. Activation of NF-jB
NF-jB is an important transcription factor that mediates
the response to infection through regulation of genes in-
volved in the immune response [56]. The major form of
NF-jB consists of p50 and the p65 (Rel A) DNA-binding
protein [57]. In its inactive form NF-jB is found in the
cytoplasm bound to inhibitor proteins, the best known of
which are IjBa and IjBb. Stimulation of cells by various
extracellular agents such as viruses, oxidants, inflammatory
cytokines and lipopolysaccharide leads to phosphorylation
of IjBa and IjBb by a phosphorylation cascade involving
specific IjB kinases and subsequent ubiquitination. Once
these proteins are degraded, NF-jB is released from the
complex and translocates into the nucleus, where it regulates
the expression of many targets including proinflammatory
cytokines.
Lm infection has been shown to activate NF-jB in a
number of different cell types. In P388D1 murine macroph-
ages a biphasic activation of NF-jB was observed. Transient
NF-jB activation was induced by adhesion of Lm to P388D1
cells, which also occurred upon addition of nonvirulent L.
innocua or by addition of listerial cell wall lipoteichoic acid
to these cells [57,58]. The second phase of NF-jB activation
was persistent and appeared to require a signal induced
during escape of Lm from the phagosome or during intracel-
lular replication, as NF-jB activation was significantly lower
when cells were infected with phospholipase mutants (plcA
or plcB) [57]. ActA expression is also required for full
NF-jB activation, possibly because the bacteria must enter
secondary vacuoles for full activation of BR-PLC [39,59].
In the Caco-2 epithelial cell line, Lm infection induced a
monophasic activation of NF-jB attributed to lipoteichoic
acid. Addition of cytochalasin D, which blocks bacterial
invasion, failed to inhibit NF-jB activation, indicating that

H. Goldfine, S.J. Wadsworth / Microbes and Infection 4 (2002)
1341
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unpublished results). Since LLO is required for escape from
the primary vacuole and the loss of PI-PLC results in de-
creased escape, the continued function of these proteins is
critical at this stage of infection. It is not clear if LLO is
needed for continued PKC signaling invoked by LLO and
phospholipase activities or for disruption of the vacuole to
allow bacterial escape.
Addition of Lm or secreted proteins outside HUVEC or
human neutrophils results in release of arachidonic acid and
the formation of eicosanoids, presumably through the activa-
tion of host phospholipase A2[44–46]. Activation of NF-jB
also occurs from outside the macrophage through binding of
Lm cell wall molecules to surface receptors [57,58]. Activa-
tion of NF-jB is biphasic. After entry, sustained activation
requires LLO to obtain release of bacteria into the cytosol,
and the expression of PI-PLC (plcA) and BR-PLC (plcB)
[57]. Presumably lipid mediators such as DAG and ceramide,
which are elevated during infection of J774 macrophages by
Lm [31], are involved, but this connection has not been
proven.
It appears that Lm manipulates intracellular signaling in
macrophages at all steps of infection including (a) the initial
phase in which bacteria interact either through secreted pro-
teins or by contact; (b) after internalization, when bacteria
reside in an endocytic vacuole; (c) upon release into the
cytosol; and (d) during cell-to-cell spread, when Lm resides
briefly in the double-membrane secondary vacuole. This
organism, which has already provided much new informa-
tion on mammalian cell biology, undoubtedly will continue
to reveal many novel facets of the inner workings of its host
cells.
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Acknowledgements
The work in the authors’ laboratory reviewed here was
supported by grants from the National Institute of Allergy
and Infectious Diseases and the National Institute of General
Medical Sciences of the United States Public Health Service.
We wish to thank Hélène Marquis, Hao Shen, Mathilde
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