1 INSERM U411, Faculté de Médecine Necker-Enfants Malades,
Université René Descartes, Paris, France
2 CNRS UPR 415, Institut Cochin de Génétique Moléculaire,
Paris, France
* Author for correspondence (e-mail: nassif{at}necker.fr
Accepted 12 December 2001
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Summary |
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Key words: Neisseria, Ezrin, Microvilli, Meningitis
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Introduction |
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Neisseria meningitidis (also referred to as meningococcus) is an
extracellular pathogen that, once in the bloodstream, invades the meninges and
the cerebrospinal fluid. A key event in N. meningitidis invasion is
the interaction of bacteria with brain endothelial cells. Indeed, examination
of post-mortem material has clearly shown a direct interaction of
extracellular N. meningitidis with endothelial cells of both the
choroid plexus and meningeal capillaries
(Pron et al., 1997). Two
meningococcal attributes are clearly essential for meningeal invasion: (i) the
capsular polysaccharide, which allows bacterial survival in extracellular
fluids, and (ii) type IV pili (TFP), which are multimeric structures essential
for adhesion of virulent capsulated N. meningitidis to host cells
(Nassif et al., 1999
). This
latter process seems to be due to a tip-located adhesin designated PilC
(Rudel et al., 1995
). In vitro
studies using tight monolayers of human epithelial cells have shown that
piliated capsulated meningococci can cross the monolayers by transcytosis
(Merz et al., 1996
;
Pujol et al., 1997
), thus
suggesting that, in vivo, meningococci may use the transcellular route to
cross the vascular endothelium to the meninges. For non-capsulated
meningococci, as well as for the closely related pathogen N.
gonorrhoeae, several mechanisms of bacterial internalization have been
described, that involve interactions of cellular receptors with bacterial
surface components, such as Opa and Opc outer membrane proteins
(Bauer et al., 1999
;
Edwards et al., 2000
;
Meyer, 1998
;
Naumann et al., 1999
;
Virji et al., 1993
;
Virji et al., 1992
;
Virji et al., 1996
). The role
of these components in virulent capsulated strains of N. meningitidis
is uncertain since non-piliated capsulated isolates are unable to efficiently
interact with the cells. In addition, piliated capsulated N.
meningitidis, which do not express Opa and Opc outer membrane proteins,
can efficiently be internalized (Merz et
al., 1996
; Pujol et al.,
1997
; Pujol et al.,
1999
). Type IV pili thus remain the only component able to
initiate the interaction of virulent capsulated N. meningitidis with
human cells.
Pilus-mediated adhesion of capsulated strains was shown to lead to the
formation of cortical plaques underneath bacterial colonies on the apical
surface of epithelial cells. These structures result from the localized
polymerization of cortical actin associated with the clustering of tyrosine
phosphorylated and integral membrane proteins (ICAM-1, CD44, EGF receptor) as
well as ezrin, a member of the ERM (ezrin-radixin-moesin) protein family
(Merz et al., 1999;
Merz and So, 1997
). The role
and the mechanisms by which these cytoskeletal modifications eventually lead
to bacterial internalization remained mostly unknown. Recently we have
observed in endothelial cells that ErbB2, a receptor-tyrosine kinase of the
EGF receptor family, is specifically recruited underneath bacterial colonies
following pilus-mediated adhesion. ErbB2 is activated by homodimer formation
and is required for an efficient bacterial internalization, whereas
ErbB2-associated signaling is not involved in bacteria-induced cytoskeletal
modifications (Hoffmann et al.,
2001
).
The ERM proteins play a major structural and regulatory role in many of the
morphogenic changes of the plasma membrane, including the formation of
microvilli (Yonemura and Tsukita,
1999). These proteins control the organization of the cortical
cytoskeleton by acting as linkers between the plasma membrane and the actin
cytoskeleton. ERM proteins interact through their amino-terminal domain with
the cytoplasmic domain of transmembrane proteins (so-called ERM binding
proteins), such as CD44 or ICAM-1, and interact with F-actin by their
C-terminal domain. ERM proteins are in a cytosolic dormant form, in which the
binding site for F-actin is masked by an intramolecular interaction between
the N- and C-terminal domains (Mangeat et
al., 1999
). Activation of ERM seems to require the binding of
phosphotidylinositol 4,5-biphosphate and the phosphorylation of a specific
threonine residue in their C-terminal domain by Rho-kinase, an effector of Rho
GTPase (Gautreau et al., 2000
;
Matsui et al., 1998
;
Matsui et al., 1999
).
In this work, we show that the internalization of piliated capsulated meningococci in endothelial cells, a process associated with meningeal invasion, is triggered by the formation of microvilli-like membrane protrusions, but not ruffles, containing ezrin, moesin and the ERM-binding proteins CD44 and ICAM-1. The role of ezrin recruitment in actin polymerization was confirmed by expression of a dominant-negative form of ezrin. Furthermore, we provide evidence that both Rho and Cdc42, but not Rac1, are critical for actin cytoskeletal modifications induced by N. meningitidis and subsequent bacterial internalization, although ERM binding to the plasma membrane occurs through a Rho-GTPase-independent pathway. All together, these data indicate that the formation of microvilli-like membrane protrusions at the surface of human endothelial cells is critically involved in the internalization of piliated capsulated virulent N. meningitidis.
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Materials and Methods |
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Cell culture and infection
Human umbilical vein endothelial cells (HUVECs; PromoCell, Heidelberg,
Germany) were used between passages 1 and 8 and grown in Endo-SFM supplemented
with 10% heat-inactivated FCS and 2 mM L-Glutamine (Life-Technologies, Grand
Island, USA), 1 ng/ml of bFGF (Boehringer-Mannheim, Meylan, France), 0.5 UI/ml
of heparin and 1.25 µg/ml of endothelial cell growth supplement (Sigma,
Saint Louis, USA). The Human bone marrow endothelial cell line (HBMECs)
(Schweitzer et al., 2000) was
kindly provided by B. Weksler (Weill Medical College of Cornell University,
NY, USA). Cells were cultured in DMEM-glutamax (Life-Technologies, Grand
Island, USA) supplemented with 10% heat inactivated FCS, 7 UI/ml of heparin,
7.5 µg/ml of endothelial cell growth supplement and 10 mM Hepes. Cells were
seeded at 5x104 cells/cm2 in a 24-well or 6-well
culture plates and grown at 37°C in a humidified incubator under 5%
CO2 for 2 to 3 days.
The day before the infection, the culture medium was replaced by a serum-free medium (starvation medium). Approximately 107 bacteria in culture medium were added to the cells and allowed to adhere for 15-30 minutes. The monolayers were then washed every hour and new medium was added to avoid reinfection from the supernatant. At the indicated times, monolayers were harvested and the number of adhesive bacteria determined.
The number of internalized bacteria was determined by a gentamicin protection assay. Cells grown in a 24-well or 6-well culture plate were infected as above. 4 and 7 hours after infection, cells were washed and 150 µg/ml of gentamicin was added to each well to kill extracellular bacteria. After 1 hour of incubation at 37°C, cells were scraped off the plates, and the number of intracellular bacteria was then determined. The proportion of internalized bacteria was calculated as the ratio of those that were gentamicin-resistant to adherent bacteria.
Where indicated, cells were pretreated with the following compounds,
diluted in starvation medium: 1 ng/ml of Clostridium difficile toxin
B (ToxB), a non-selective inhibitor of the Rho family GTPases
(Boquet, 1999), kindly provided
by P. Boquet (INSERM, Nice, France) or 30 µM of Y27632, a selective
inhibitor of Rho kinase (Maekawa et al.,
1999
), an effector of the Rho protein, a gift from Yoshitomi
Pharmaceutical (Osaka, Japan). These treatments were initiated 16 hours or 1
hour respectively, prior to the addition of bacteria and were maintained
during the course of the experiments.
Antibodies
Anti-human CD44 mAb was purchased from R&D (Abington, UK), anti-ICAM-1,
anti-VE-cadherin mAbs were from Serotec (Oxford, England), anti-paxillin mAb
was from Transduction Laboratories (Lexington, USA) and anti-human vinculin
mAb from Sigma (Saint Louis, USA). Phalloidin-alexa488 (Molecular Probes,
Eugene, Or) was used to stain the actin cytoskeleton (F-actin). ERM proteins
were detected using selective rabbit polyclonal antisera kindly provided by P.
Mangeat (CNRS, Montpellier, France). mAb 26C4 specifically raised against RhoA
(Lang et al., 1993) is a gift
from J. Bertoglio (INSERM, Chatenay-Malabry, France). In infected monolayers,
bacteria were stained using either ethidium bromide or a rabbit polyclonal
antibody directed against the meningococcus strain Rou. This antibody was
generated as follows: an overnight culture of Rou was either fixed with 2.5%
paraformaldehyde or sonicated. These preparations were mixed with Freund's
complete adjuvant (1:1), and 1 ml was used for inoculation of a New Zealand
white rabbit. The rabbit was boosted with the same mixture three times every 2
weeks. Microinjected proteins were detected by immunostaining using antimyc
9E10 mAb from Biomol (Plymouth, USA) or anti-VSVG P5D4 mAb from Roche
(Indianapolis, USA).
Immunofluorescence protocol and confocal microscopy
HUVECs were plated at a density of 5x104
cells/cm2 onto 12-mm diameter acid-washed glass coverslips coated
overnight with fibronectin (10 µg/ml). Cells were infected as described
above, fixed with 2.5% paraformaldehyde in PBS for 20 minutes, neutralized
with 0.1 M glycine in PBS for 5 minutes, permeabilized for 1 minute with 0.5%
Triton X100 in PBS and then saturated for 20 minutes with PBS containing 0.2%
gelatin before incubation for 30 minutes with the primary antibodies diluted
in PBS/gelatin. Cells were then washed with PBS and incubated for 30 minutes
with anti-rabbit or anti-mouse secondary antibodies conjugated with Cy3 or Cy5
and immunoabsorbed against human, bovine, rabbit or mouse serum proteins
(Jackson Immunoresearch, West Grove, USA). Finally, cells were washed three
times in PBS and mounted in moviol (Sigma, Saint Louis, USA) before analysis
with a Zeiss LSM 510 confocal microscope. The pinhole of each PMT was adjusted
separately in order to take XY optical sections with the same step. Sequential
scannings were also performed in order to confirm the absence of any
interference between signals detected at different wavelengths. Series were
subjected to an orthogonal projection in order to make a two-dimensional
reconstruction of the area under study.
Microinjection
Microinjections were performed using an Eppendorf Transjector 5246 coupled
to an Eppendorf Micromanipulator 5179. HUVECs were seeded onto glass
coverslips as described for the immunofluorescence protocol. About 50 cells
per well were microinjected into the nucleus with the eukaryotic expression
vector. The vector used was pRK5-myc, encoding the dominant-negative forms of
Rac or Cdc42, RacN17 or Cdc42N17
(Nobes and Hall, 1999). These
plasmids were kindly provided by G.Tran Vhan Nieu (Institut Pasteur, Paris).
pRK5-myc-RacN17 was microinjected at 117 µg/ml 18 hours before
cell infection and pRK5-myc-Cdc42N17 at 133 µg/ml 5 hours before
cell infection. Eukaryotic expression vectors encoding either the N- terminal
domain of human ezrin (amino acids 1-309), a dominant-negative form of ezrin
(pcB6-ezrin-Nter) or the full-length ezrin (pCB6-Ezrin) were obtained from A.
Gautreau (Institut Curie, Paris). They were microinjected into the nuclei of
HUVECs at a concentration of 120 µg/ml 2 hours prior infection of the
monolayers with N. meningitidis.
The recombinant C3-transferase from Clostridium botulinum
(Boquet, 1999), a Rho-selective
inhibitor generously provided by M. Popoff (Institut Pasteur, Paris) was
injected into the cytoplasm at 200 ng/ml with 2.5 mg/ml rhodamine-labelled
dextran diluted in distilled water, 2 hours before infection by N.
meningitidis as described above. Control injections carried out with
rhodamine-labelled dextran did not produce any significant effect on cell
morphology or actin organization.
Electron microscopy
For scanning electron microscopy, cells were cultured and infected as
described above, then fixed with 2.5% glutaraldehyde solution in 0.1 M
cacodylate buffer pH 7.3 Preparations were then coated with gold palladium
after critical-point drying. The examination was performed on a JEOL 840A at
the Centre Inter-Universitaire de Microscopie Electronique (Paris,
France).
For transmission electron microscopy, cells were grown on transwells as described above and were fixed overnight at 4°C with a 1:1 mixture of 2.5% glutaraldehyde and 2.5% paraformaldehyde in cacodylate sucrose buffer (0.1 M cacodylate, 0.1 M sucrose, 5 mM CaCl2, 5 mM MgCl2, pH 7.2). Monolayers were then stained for 1 hour in a solution of 1% OsO4 and placed for 1 hour in 1% uranyl acetate. After dehydration in a graded series of alcohols, cells were embedded with polyester filter in Epon. Thin sections were obtained by using an Ultracut ultramicrotome and analyzed with JEOL-100CX electron microscope.
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Results |
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Prior to bacterial infection, no membrane protrusion was observed on
endothelial cells, whereas during localized adhesion of N.
meningitidis, small protrusions of the cellular membranes were induced
beneath and around bacteria of most colonies
(Fig. 1A,B). The formation of
these membrane protrusions was transient and disappeared during the stage of
diffuse adhesion (Fig. 1C,D).
At this later time point, even though bacteria remained highly adhesive to the
endothelial cell surface, no intimate adhesion with pedestal formation was
observed with endothelial cells, in contrast to our previous observations with
infected epithelial cells (Pujol et al.,
1999). Moreover, as shown in
Fig. 1, when interacting with
endothelial cells, meningococci remained mostly extracellular. However, in
some cases, cellular protrusions can be seen surrounding and engulfing
meningococci (Fig. 1B).
Transmission electron microscopy analysis
(Fig. 2A-D) confirmed that
bacteria can be found surrounded by such membrane protrusions, leading to the
formation of large vacuoles containing meningococci. These observations
strongly suggest that these protrusions are responsible for meningococcal
internalization
|
To confirm that bacterial internalization is correlated with the formation of these cellular protrusions induced during the localized adhesion of N. meningitidis, gentamicin protection assays were performed at 4 hours (localized adhesion) and 8 hours (diffuse adhesion) after infection of the endothelial monolayers. As shown in Fig. 3, whereas the number of adhering bacteria dramatically increased between 4 hours and 8 hours, owing to extracellular proliferation, the number of internalized bacteria remained relatively unchanged during the same time, thus indicating that bacterial internalization predominantly takes place during the localized adhesion, when cellular protrusions are observed. Taken together, these observations suggest that piliated capsulated meningococci induce the transient formation of cellular protrusions during the initial adhesion phase of N. meningitidis infection and that these cytoskeletal modifications are associated with the internalization of a small fraction of adhesive bacteria. Ezrin is required for the assembly of the microvilli-like cellular protrusions
|
In order to gain insight into the molecular mechanisms responsible for the formation of the cellular protrusions induced by pilus-mediated adhesion of N. meningitidis, we identified components involved in their organization. Since the cortical actin-based cytoskeleton plays a crucial role in local membrane reorganization, we assessed, by immunofluorescence analysis, whether actin was polymerized in the membrane protrusions induced by N. meningitidis. As shown in Fig. 4A, localized polymerization of cortical actin was observed underneath bacterial colonies. Moreover, xz section analysis of the actin staining (Fig. 4D) revealed that cortical actin was indeed polymerized in the membrane protrusions.
|
We then analyzed the distribution of various proteins known to link the
actin cytoskeleton and membrane components. Paxillin
(Fig. 4B) and vinculin (data
not shown) were not recruited to the site of polymerization of cortical actin
beneath bacterial colonies but remained highly enriched at the tip of actin
stress fibers in focal adhesions, indicating that cytoskeletal modifications
induced by N. meningitidis differed from focal adhesions or focal
complexes that are seen in ruffles (Yamada
and Geiger, 1997; Zamir et
al., 1999
). On the other hand, using specific antibodies, ezrin
(Fig. 4C) and moesin (not
shown) were found to be recruited underneath bacterial colonies to the site of
actin polymerization together with the ERM-binding proteins CD44
(Fig. 4C) and ICAM-1 (not
shown), whereas radixin, another member of the ERM family, remained localized
at cell junctions (not shown). In addition, xz section analysis of ezrin
distribution in infected cells showed a massive recruitment just beneath the
plasma membrane at the tip of the cellular protrusions
(Fig. 4D), reminiscent of ezrin
distribution in epithelial microvilli
(Yonemura and Tsukita,
1999
).
The ERM N-terminal domain interacts with ERM-binding proteins whereas the C-terminal domain binds to F-actin. Indeed, the N-terminal domain (amino acids 1-309) of ezrin can compete with ezrin for interaction with ERM-binding proteins, thus behaving as a dominant-negative form of ezrin. In order to assess the actual function of ezrin in the assembly of the bacteria-induced cellular protrusions, we expressed its N-terminal domain in endothelial cells before N. meningitidis infection. As shown Fig. 5A, expression of this dominant-negative form of ezrin not only inhibited the recruitment of endogenous ezrin but also prevented polymerization of actin underneath bacterial colonies. This observation was further confirmed by cell counting (Fig. 5B). It should be pointed out that microinjection of full-length ezrin had no effect on actin polymerization as shown in Fig. 5C, thus eliminating the possibility that the lack of actin polymerization observed in Fig. 5B was a consequence of microinjection. These data clearly indicate that ezrin is playing a pivotal role in the cytoskeletal modifications induced by pilus-mediated adhesion of N. meningitidis.
|
Clostridium difficile toxin B inhibits cortical actin
polymerization but not ezrin recruitment underneath bacterial colonies
To investigate the putative role of Rho family GTPases in the organization
of membrane protrusions induced by N. meningitidis, endothelial cell
monolayers were incubated with Clostridium difficile toxin B (Tox B),
which non-selectively inhibits all members of the Rho family
(Boquet, 1999). The number of
bacterial colonies inducing cortical actin polymerization and/or ezrin
recruitment was measured (Fig. 6A and
6B). Tox B induced a dramatic decrease in the number of colonies
associated with cortical actin polymerization
(Fig. 6B). However, the number
of bacterial colonies recruiting ezrin was not affected
(Fig. 6A), although, as shown
in Fig. 6C, ezrin recruitment
was less pronounced than that observed with no ToxB treatment when actin was
polymerized. We then conclude that Rho family GTPases are involved in cortical
actin polymerization but not in the initial ezrin and ezrin-binding protein
recruitment induced by N. meningitidis underneath bacterial
colonies.
|
Cdc42 and Rho are required for actin polymerization induced by N.
meningitidis and bacterial internalization
We next determined which GTPases of the Rho family were involved in the
cytoskeletal modifications induced by N. meningitidis. For that
purpose, we used a monoclonal antibody specifically raised against RhoA, 26C4
(Lang et al., 1997). Immunofluorescence analysis of RhoA distribution in
infected cells demonstrated an enrichment of this molecule in the
microvilli-like structures (Fig.
7A). Furthermore, the role of Rho was further analyzed using (i)
Clostridium botulinum C3 transferase, a selective inhibitor of Rho,
and (ii) Y27632, a selective inhibitor of Rho kinase, a Rho effector
(Maekawa et al., 1999). Under
the conditions used, Y27632 induced a loss of actin stress fibers, without
inducing cell rounding, and prevented actin polymerization underneath
bacterial colonies (Fig. 7B).
Indeed, the number of bacterial colonies able to induce cortical actin
polymerization was significantly lower in cells incubated with Y27632 than in
control cells (Fig. 8). Similar
results were obtained after microinjection of C3 transferase
(Fig. 8). All together, these
data demonstrate the role of Rho and of its effector Rho kinase in mediating
cortical actin polymerization induced by N. meningitidis.
|
|
We next investigated whether the related proteins, Rac1 and Cdc42, also
affected N. meningitidis-induced actin polymerization. HUVECs were
microinjected with plasmids encoding Rac1N17 and
Cdc42N17, the dominant-negative forms of Rac1 and Cdc42,
respectively. As shown in Fig.
8, the number of bacterial colonies with polymerized actin was
significantly reduced in endothelial cells microinjected with
Cdc42N17, whereas microinjection of Rac1N17 had no
effect. Fig. 7C shows that
microinjection of Cdc42N17 induced a dramatic increase in the
formation of stress fibers, a cytoskeletal organization controlled by Rho
(Hall, 1998), without inducing
polymerization of cortical actin underneath bacterial colonies (see xz section
in Fig. 7C). This increase in
the formation of stress fibers in cells microinjected with a dominant-negative
form of Cdc42 indicates that the effect of Cdc42 N17 on actin
cytoskeleton is not due to an inhibition of Rho activity.
Fig. 7D confirmed that
microinjection of the dominant-negative form of Rac1 had no effect on cortical
actin polymerization induced by N. meningitidis, even though
microinjected cells were retracted and had no stress fibers, as expected from
Rac1 inhibition (Hall, 1998
).
Taken together, these data demonstrate that the cytoskeletal modifications
induced by N. meningitidis required the activation of both Cdc42 and
Rho but not of Rac1.
To confirm that bacterial internalization is promoted by the induction of these cytoskeleton changes, we analyzed the effect of ToxB and Y27632 on bacterial adhesion and entry into endothelial cells. As shown in Fig. 9, bacterial entry into cells treated by either of these two inhibitors was significantly decreased, whereas N. meningitidis adhesion was unaffected. These data demonstrate that actin cytoskeleton reorganization induced by both Cdc42 and Rho, via Rho kinase activation, are crucial for the internalization of N. meningitidis into endothelial cells. They suggest that in vivo activation of both Cdc42 and Rho during pilus-mediated adhesion onto brain endothelial cells may be involved in meningeal invasion by N. meningitidis.
|
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Discussion |
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The precise mechanism by which pilus-mediated adhesion is recruiting ezrin
is still unknown. ERM proteins have been shown to be present in the cytoplasm
as oligomers, which can be recruited to the cytoplasmic membrane where
threonine phosphorylation in the C-terminal domain is required for the
transition to monomers. Only monomers can crosslink actin filaments to the
plasma membrane to form microvilli in epithelial cells
(Gautreau et al., 2000). The
N-terminal domain is responsible for membrane targeting via interaction with
ezrin-binding proteins, such as CD44 or ICAM-1. Indeed, CD44 has been proposed
to be involved in the formation of microvilli by concentrating activated ERM
proteins at the plasma membrane (Yonemura
et al., 1998
; Yonemura and
Tsukita, 1999
). We therefore hypothesize that, during
pilus-mediated adhesion of N. meningitidis, the recruitment of
ezrin-binding proteins might similarly target ezrin and moesin to the plasma
membrane, underneath bacterial colonies, where they would be activated and
would trigger actin polymerization.
Several studies have shown that Rho stimulates the activity of ezrin and
moesin (i) by inducing their recruitment to the plasma membrane, (ii) by
promoting the formation of phosphotidylinositol 4,5-biphosphate, which is
responsible for an activating conformational change in ERM proteins, and
(iii), although still a matter of debate
(Matsui et al., 1998;
Matsui et al., 1999
), by
activating Rho kinase, which can phosphorylate ERM proteins in their
C-terminal domain. Surprisingly, we observed in the present study that ToxB, a
non-selective inhibitor of Rho family GTPases, did not affect the recruitment
of ERM proteins or ERM-binding proteins underneath bacterial colonies. Further
investigation will aim at identifying the alternative signaling pathways
involved in ERM protein recruitment promoted by the pilus-mediated adhesion of
N. meningitidis.
Our data using specific inhibitors of the Rho family GTPases demonstrate
the crucial role of both Rho and Cdc42 in N. meningitidis-induced
actin polymerization and bacterial entry. However, their respective
contribution still remains unknown, as does the identity of Cdc42 targets.
Considering the role of Rho kinase in actin polymerization induced by N.
meningitidis, it is tempting to speculate that the role of Rho activation
is to promote ezrin phosphorylation following its recruitment to the plasma
membrane at the site of bacterial attachment. Since a role for Cdc42 in the
formation of microvilli-like structures has previously been reported in
epithelial cells (Gauthier-Rouviere et al.,
1998), we hypothesize here that it might be responsible for actin
nucleation leading to the formation of the microvilli-like membrane
protrusions in endothelial cells.
Interestingly, the membrane protrusions induced by N. meningitidis
on the endothelial surface significantly differ from those induced by other
known invasive bacteria. Cytoskeletal modifications leading to the formation
of ruffles have been shown to be responsible for the internalization of
bacterial pathogens such as Shigella, Salmonella and, more recently,
Neisseria gonorrhoeae (Edwards et
al., 2000; Galan and Zhou,
2000
; Nhieu and Sansonetti,
1999
). In the case of Shigella, ezrin recruitment was
shown to be dependent on bacteria-induced actin polymerization
(Skoudy et al., 1999
).
Moreover, Shigella-induced protrusions are enriched in paxillin and
vinculin, two proteins usually localized at focal adhesions, which are not
recruited in N. meningitidis-induced membrane protrusions. Indeed,
the lack of ruffle formation reported in this study in response to N.
meningitidis adhesion was correlated with our observation that Rac1,
which generally controls ruffle formation
(Hall, 1998
), is not involved
in bacteria-induced actin cytoskeleton modifications in endothelial cells.
Despite the lack of the type III secretion system, which is involved in
Shigella and Salmonella internalization, virulent capsulated N.
meningitidis are internalized into host cells. This internalization is
likely to promote their subsequent transcytosis through cellular barriers
in vivo. As previously mentioned, type IV pili are the only bacterial
components identified so far involved in the formation of the microvilli-like
membrane protrusions induced by N. meningitidis. That pilusmediated
adhesion has been shown to be important for meningeal invasion
(Pron et al., 1997) is
consistent with the role of these cytoskeletal modifications in the crossing
of the cerebral vascular endothelium. It is, however, possible that additional
meningococcal components are involved in the signaling events leading to the
formation of the membrane protrusions observed here. Recently, a role for the
lipooligosaccharide (LOS) in the actin cytoskeleton rearrangement induced by
piliated Neisseria gonorrhoeae has been shown
(Song et al., 2000
).
Gonococcal strains expressing LOS lacking the lacto-N-neotetraose did not
promote actin polymerization and were less invasive. However, we did not
observe here such inhibition of actin polymerization with a mutant of the ROU
strain lacking the LOS core (data not shown).
The present study thus provides new insights into the molecular mechanisms used by N. meningtidis to enter endothelial cells, a critical step in the pathogenesis of this bacteria for its trafficking through the cerebral vasculature. Further studies on the meninogoccal components and their cellular receptors involved in this interaction should provide essential clues to the understanding of the molecular mechanisms of N. meningitidis infection of the central nervous system.
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Acknowledgments |
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