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 Progress in Neurobiology 91 (2010) 130139

Contents lists available at ScienceDirect

Progress in Neurobiology
journal homepage: www.elsevier.com/locate/pneurobio

Mechanisms of meningeal invasion by a bacterial extracellular pathogen, the

example of Neisseria meningitidis
Olivier Join-Lambert a,b,d,1,2, Philippe C. Morand a,b,d,1,2, Etienne Carbonnelle a,b,d,1,2, Mathieu Coureuil a,d,1,
Emmanuelle Bille a,b,d,1,2, Sandrine Bourdoulous c,d,3, Xavier Nassif a,b,d,*
a
INSERM, Unite 570, 156 rue de Vaugirard, 75015 Paris, France
b
Service de Microbiologie, Assistance-Publique - Hopitaux de Paris, Hopital Necker-Enfants Malades, 149 rue de Sevres, 75015 Paris, France
c
Institut Cochin, Departement de Biologie Cellulaire et des Interactions Hotes Pathogenes, INSERM, Unite 567, CNRS, UMR8104, 22 rue Mechain, 75014 Paris, France
d
Universite Paris Descartes, Paris, France

A R T I C L E I N F O A B S T R A C T

Article history: The bloodcerebrospinal uid (CSF) barrier physiologically protects the meningeal spaces from
Received 13 May 2009 bloodborne bacterial pathogens, due to the existence of specialized junctional interendothelial
Received in revised form 8 September 2009 complexes. A few bacterial pathogens are able to reach the subarachnoidal space and cause bacterial
Accepted 10 December 2009
meningitis in humans, a rare but dreadful disease. Surprisingly, most of them are extracellular
commensals of the nasopharynx (Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus
Keywords: inuenzae) or of the digestive tract (Escherichia coli and Streptococcus agalactiae). The particular ability of
Central nervous system infections
these pathogens to induce meningitis is related to virulence factors that allow them to escape host innate
Bloodbrain barrier
Endothelial cells
immunity, to multiply within the serum, and to interact closely with the endothelial front line of defense
Tight junctions of the bloodCSF barrier. In vitro studies using microvascular brain endothelial cell lines have shown that
Neisseria meningitidis induced transcytosis may be a common route used by H. inuenzae, S. pneumoniae, E. coli and S. agalactiae
to reach the CSF. N. meningitidis is a strict human pathogen that interacts very tightly with endothelial
cells. Adhesion of the meningococcus is mediated by type IV pili that induce a localized remodeling of the
sub cortical cytoskeleton, leading to the formation of endothelial membrane protrusions that anchor
bacterial colonies at the endoluminal face of the endothelial cell membrane, allowing a better resistance
to blood ow. Recent work has shown that N. meningitidis is also able to recruit the polarity complex
Par3/Par6/aPKC that re-routes endothelial cell adhesion molecules of interendothelial junctions,
opening a paracellular route for bacteria to cross the endothelial barrier.
2009 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............... .............. . . . . . . 131

2. Structural and functional heterogeneity of the bloodCNS interfaces. . . . . . . . . . . . . . . . . . . . . ............... .............. . . . . . . 131
2.1. The bloodbrain barriers sensu stricto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............... .............. . . . . . . 131
2.2. The bloodCSF barriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............... .............. . . . . . . 132
3. Crossing of the bloodCSF barriers by extracellular bacterial pathogens . . . . . . . . . . . . . . . . . . ............... .............. . . . . . . 132
3.1. Where is the bloodCSF barrier crossed? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............... .............. . . . . . . 132
3.2. How do extracellular bacteria break the bloodCSF barrier? . . . . . . . . . . . . . . . . . . . . . . ............... .............. . . . . . . 134
3.3. Role of cytokines and innate immunity in bacterial entry into the CNS . . . . . . . . . . . . . ............... .............. . . . . . . 134
4. The example of cerebrospinal meningitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............... .............. . . . . . . 135
4.1. Type IV pili are the major meningococcal attributes allowing blood-carried bacteria to interact with CNS endothelial cells . . . . . . 136

Abbreviations: CNS, central nervous system; CSF, cerebrospinal uid; BBB, bloodbrain barrier; CP, choroid plexus; TfP, type IV pili.
* Corresponding author at: INSERM, Unite 570, 156 rue de Vaugirard, 75015 Paris, France. Tel.: +33 1 40 61 53 79; fax: +33 1 40 61 56 77.
E-mail addresses: olivier.join-lambert@inserm.fr (O. Join-Lambert), philippe.morand@inserm.fr (P.C. Morand), etienne.carbonnelle@egp.aphp.fr (E. Carbonnelle),
mathieu.coureuil@inserm.fr (M. Coureuil), emmanuelle.bille@inserm.fr (E. Bille), sandrine.bourdoulous@inserm.fr (S. Bourdoulous), xavier.nassif@inserm.fr (X. Nassif).
1
Tel.: +33 1 40 61 53 79; fax: +33 1 40 61 56 77.
2
Tel.: +33 1 44 49 49 61; fax: +33 1 44 49 49 60.
3
Tel.: +33 1 40 51 64 27; fax: +33 1 40 51 64 30.

0301-0082/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pneurobio.2009.12.004
 O. Join-Lambert et al. / Progress in Neurobiology 91 (2010) 130139 131

4.2. Signaling triggered by N. meningitidis that leads to the extravasation of brain vessels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
4.2.1. Tight adhesion and microcolony formation at the endothelium surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
4.2.2. Adherent bacteria induce an intracellular signaling that leads to a localized cytoskeleton remodeling of endothelial cells 136
4.2.3. N. meningitidis opens a paracellular route to cross the endothelial bloodCSF barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

1. Introduction In this review, using N. meningitidis as a model we address the

Bacterial meningitis is the leading cause of central nervous
system (CNS) infections. Although bacterial meningitis can be due
to the dissemination of contiguous infections such as sinusitis or
mastoiditis to the meningeal membranes, most cases are caused by
bloodborne pathogens. The bloodbrain barrier (BBB) protects the
CNS from most bacteria that may have reached the bloodstream,
thus restricting the etiology of bacterial meningitis to a few and
predominantly extracellular pathogens: Escherichia coli K1 and
Streptococcus agalactiae (Group B Streptococcus) in the newborn,
Neisseria meningitidis, Haemophilus inuenzae type b and Strepto-
coccus pneumoniae in children and adults (Huang et al., 2000; Pong
and Bradley, 1999; Van de Beek et al., 2006). Paradoxically, these
bacteria are commensal of the nasopharynx (N. meningitidis, S.
pneumoniae and H. inuenzae) or of the digestive tract (E. coli and S.
agalactiae) (Nassif et al., 2002).
The pathophysiology of bacterial meningitis is a multistep
process that reects the ability of bacterial pathogens to cross the
oropharyngeal or digestive mucosal barrier, survive and replicate
in the bloodstream, and to eventually cross the bloodcerebro-
spinal uid (CSF) barrier (Nassif et al., 2002; Quagliarello and
Scheld, 1992; Rubin et al., 1985). The small number of bacterial
species capable of invading the meninges suggests that specic
virulence factors are required for bacteria to enter the subarach-
noidal space. It has been demonstrated that these extracellular
pathogens express various virulence factors allowing them to
survive in the extracellular compartments and to interact directly
with the components of the bloodbrain barrier. The main
virulence factor expressed by all extracellular pathogens is a
capsule that prevents bacterial phagocytosis or complement
mediated lysis (Austrian, 1981; Craven et al., 1980; Harrison et al.,
2002; Koedel et al., 2002; Moxon and Vaughn, 1981; Nassif et al.,
2002; Winkelstein and Moxon, 1992). These bacteria show
different propensities for entering the subarachnoidal spaces
since it has been estimated that respectively 63% and 9% of
bacteraemia due to N. meningitidis and S. pneumoniae are
associated with meningitis (InVS, 2009). Once inside the CSF,
bacterial multiplication is thought to be uncontrolled, due to the
local deciency in complement and immunoglobulins and despite
the inux of polymorphonuclear leukocytes induced by the local
inammatory response. However data obtained in primates
showed that bacterial presence in the CSF can be transient if
bacteremia is not sustained, reecting the fact that bacterial entry
into the CSF may not always lead to meningitis (Smith et al., 1982).
In contrast to intracellular pathogens such as Listeria mono-
cytogenes that cause both meningitis and encephalitis (Drevets
et al., 2004; Vazquez-Boland et al., 2001), extracellular bacterial
pathogens are responsible for meningitis only, thus suggesting
that these bacteria are unable to successfully cross the
parenchymal bloodbrain barrier, and/or to induce an inamma-
tion, and/or to multiply in the brain parenchyma. Severe medical
complications of bacterial meningitis such as cerebral edema and
vasculitis occur in 2030% of patients despite appropriate
antimicrobial treatments. These complications mainly result
from the local inammatory response, rather than bacterial
invasion of the brain parenchyma (Koedel et al., 2002; Pster et al.,
1994; Van de Beek et al., 2004).
various mechanisms of bacterial host interaction that can lead to
the crossing of the bloodCSF barrier and the subsequent
meningeal invasion.
2. Structural and functional heterogeneity of the bloodCNS
interfaces
2.1. The bloodbrain barriers sensu stricto
The bloodbrain barrier stricto senso is a highly specialized
structural and functional component of the central nervous system
that separates the circulating blood from the brain and spinal cord
parenchyma. Among the different cellular types that make up the
BBB, endothelial cells form the front defense line of the CNS
parenchyma against invading pathogens and most research on this
topic has focused on the interaction between extracellular
pathogens and brain endothelial cell lines. Schematically, the
capillaries of the CNS parenchyma have two specic features that
are not shared by those of other peripheral organs: (i) the presence
of specialized junctional complexes, and (ii) a sparse pinocytotic
vesicular transport activity that is counterbalanced by highly
specialized transport systems that limit the entry of neuroactive
bloodborne molecules (Zlokovic, 2008). Junctional complexes are
composed of adherens junctions and of multistranded belts of tight
junctions that result in apparent membrane fusion of adjacent
endothelial cells, forming a continuous blood vessel (Fig. 1).
Interendothelial tight junctions exclude the paracellular passage of
hydrophilic macromolecules between the blood and the brain and
account for the high endothelial electric resistance of brain
capillaries (Butt et al., 1990; Crone and Christensen, 1981; Staddon
and Rubin, 1996). In vitro, the specialization of brain capillary tight
junctions has been shown to be under the control of paracrine
factors produced by astrocytic end-feet ensheathing brain
capillaries (Fig. 2A-1) (Arthur et al., 1987). At this level, the
perivascular space localized between endothelial cells and
astrocytic processes is virtual and basal lamina produced by
astrocytes and by endothelial cells are in very close contact and can
be fused. For this reason, the bloodbrain barrier at the capillary
level is also called the gliovascular or neurovascular unit.
Besides, astrocytes end-feet that form the primitive bloodbrain
barrier in non-mammalian organisms (Abbott, 2005) are also a
second line of defense that protects the brain parenchyma from the
perivascular spaces. At the periphery of the cortex, they constitute
the glia limitans that borders the brain parenchyma (Fig. 2B-2)
(Bechmann et al., 2007). Astrocytes are involved in the immune
surveillance of the perivascular spaces in various manners. They
control the entry of T lymphocytes in the neuropile by inducing
their apoptosis (Bechmann et al., 2002) and they can be activated
via numerous toll-like receptors in response to diverse stimuli,
including bacterial pathogens, thus producing proinammatory
mediators (Bechmann et al., 2007; Kadurugamuwa et al., 2005).
Other perivascular cells including pericytes, brain macrophages
and microglial cells form a perivascular immunological barrier of
the CNS that probably condition the fate of bacteria that may have
crossed a bloodCNS interface (Broadwell and Sofroniew, 1993;
Guillemin and Brew, 2004; Soulet and Rivest, 2008; Thomas, 1999).
Thus, the gliovascular unit is a dynamic structure whose main
132 O. Join-Lambert et al. / Progress in Neurobiology 91 (2010) 130139

Fig. 1. Schematic architecture of CNS interendothelial junctional complexes. CNS capillaries form a specialized continuous endothelial barrier to blood components due to the
presence of high level resistance intercellular multistranded tight junction complexes localized at the apical end of the interendothelial space. Claudin, occludin and the
junctional adhesion molecule (JAM) are the main components of tight junctions. These transmembrane proteins interact in the paracellular space in a homophilic manner,
while their intracellular domains are anchored to cytoplasmic proteins such as zonula occludens 1, 2 and 3, catenins and vinculin that interact in turn with the cellular actin
cytoskeleton (F-actin). Interendothelial junctional complexes also comprise adherens junctions that are localized at the basolateral endothelial cell membrane and stabilize
tight junctions. VE-cadherin, a member of the calcium dependent adhesion molecules is the main component of adherens junctions. Like tight junctional proteins, the
extracellular domains of VE-cadherin is linked by homophilic interactions and its intracellular domains is associated with the actin cytoskeleton via catenins and vinculin.

function is to actively regulate brain homeostasis and to protect
the brain from circulating bloodborne insults. This highly
specialized structure seems to be particularly efcient to prevent
the entry of bacteria into the brain parenchyma.
Brain post-capillary venules, venules and veins are also part of
the bloodCNS interface (Fig. 2A-2). The structure and function
of the brain venous network strikingly differs from that of brain
capillaries. It had been shown as early as the end of the 19th
century that injected dyes that did not cross the capillary blood
brain barrier were nevertheless observed in the perivascular
space of brain venules and veins. Electron microscopy studies
showed later that when venules turn into veins and exit the brain
parenchyma the structure of interendothelial tight junctions is
leakier (Ge et al., 2005; Nagy et al., 1984; Simionescu et al., 1975).
It is not known whether a paracellular or transcellular diffusion
mechanism accounts for these observations. A possible explana-
tion is that astrocytic end-feet require intimate interactions with
endothelial cells to induce a full BBB phenotype (Arthur et al.,
1987; Bechmann et al., 2007). Indeed, when capillaries turn into
venules, endothelial cells of the brain draining system are
progressively separated from astrocytic end-feet by the
VirchowRobin perivascular spaces (Fig. 2A-2). The distance
between endothelial cells and astrocytes is even more important
when veins exit the brain parenchyma in the subpial space
(Fig. 2B-2).
The structure of brain post-capillary venules and veins and their
ability to express leukocyte adhesion molecules (Cotran et al.,
1999; Ransohoff et al., 2003) likely reect their role in mediating
the entry of leukocytes and plasmatic molecules in the perivascular
space during inammatory processes (Bechmann et al., 2001,
2007). These ndings highlight that, probably due to different
physiological functions, both the anatomy and the structure of
these brains vessels differ, and that the venous system of the CNS
can be considered as a relatively vulnerable localization of the
bloodbrain barrier.
2.2. The bloodCSF barriers
The difference of composition between the blood and the CSF
reects the existence of a bloodCSF barrier that, as described for
the bloodbrain barrier, restricts the entry of blood components
into the subarachnoid space. The CSF is actively produced in brain
ventricles by the ependymal cells of the choroid plexus (CPs). The
CSF is then drained to the subarachnoidal space and resorbed
either into the blood through arachnoidal villi of the dura mater
venous sinuses, or into the cervical lymphatic system via
prolongations of the subarachnoidal space around the olfactory
nerves (Koh et al., 2005). Two localizations of the bloodCSF barrier
can therefore be distinguished: the brain ventricles and the
meningeal spaces (Fig. 2B).
The CPs form the ventricular bloodCSF barrier (Fig. 2B-1). CPs
are highly vascularized villus structures localized in brain
ventricles. As opposed to the vasculature of the CNS, the capillaries
and venules of the CPs are naturally fenestrated and lack tight
junctions, allowing a passive diffusion of serum into the CPs
stroma. The ependymal epithelial-like cells that delineate the CPs
actively produce the CSF. These cells are linked together by
junctional complexes with tight junctions, thus forming the
ventricular bloodCSF barrier.
The meningeal bloodCSF barrier (Fig. 2B-2) can be subdivided
in two compartments: the subpial space that is contiguous to the
brain, and the subarachnoidal space where the CSF circulates
within the meninges. As described above for the brain venous
system, interendothelial tight junctions of subpial and meningeal
veins form a leaky structure compared to that of brain capillaries
(Broadwell and Sofroniew, 1993). It is not known whether the pia
mater and arachnoid form a signicant barrier to limit the spread
of infection by bacteria that may have crossed the endothelial cells
barrier. Indeed, leptomeningeal cells form a thin monolayer
structure that lacks tight junctions. In patients who died of
purulent meningitis inammatory cells are present in the
subarachnoidal spaces and in the contiguous subpial space,
suggesting that inammatory cells can cross the pia mater
(Hutchings and Weller, 1986).
3. Crossing of the bloodCSF barriers by extracellular bacterial
pathogens
3.1. Where is the bloodCSF barrier crossed?
Experimental models of bloodborne meningitis with E. coli
(Moxon and Ostrow, 1977), S. agalactiae (Doran et al., 2005), H.
inuenzae (Moxon and Ostrow, 1977; Scolea et al., 1985), S.
pneumoniae (Koedel et al., 2002) and more recently N. meningitidis
(Zarantonelli et al., 2007, 2008) have been developed mostly to
identify bacterial virulence factors and to study the mechanisms of
 O. Join-Lambert et al. / Progress in Neurobiology 91 (2010) 130139 133

Fig. 2. Anatomy of bloodCNS barriers. (A) Capillary and venous bloodCNS barriers. (A1) The capillary bloodbrain barrier. Within the brain parenchyma, capillaries (blue
color) are ensheathed by astrocytic end-feet (orange color) that form the so-called glia limitans (see B2). Astrocytes not only control the paracellular permeability of the
endothelial barrier (Arthur et al., 1987; Wosik et al., 2007), but can also be activated by infectious ligands and cytokines, thus probably playing an important role in CNS
inammatory processes. At this level, between endothelial cells and astrocytic processes, both basal lamina produced by astrocytes and by endothelial cells can be fused and
the perivascular space is virtual. Other perivascular cells include pericytes, perivascular macrophages and microglial cells. They play an important role in the immune
surveillance of the CNS and probably condition the fate of bacteria that may have crossed the endothelial interface (Broadwell and Sofroniew, 1993; Guillemin and Brew,
2004; Soulet and Rivest, 2008; Thomas, 1999). At the level of CNS capillaries, the bloodbrain barrier is thus a highly specialized structure that is particularly efcient to
prevent the entry of extracellular bacteria into the brain parenchyma. (A2) The venous bloodbrain barrier. Brain post-capillary venules and veins (blue color) also form
another specialized vascular network interface of the bloodbrain barrier that differs in structure and function compared to brain capillaries. In contrast with capillary
endothelial cells, brain venules and veins interendothelial junctional complexes are more leaky. Indeed, at this anatomical level, the perivascular space (VirchowRobin
perivascular space) is not anymore virtual and endothelial cells are not in close contact with the glia limitans (orange color). The particular structure of brain venules and vein
probably reects their function that allows bloodborne leukocytes to cross the bloodbrain barrier both physiologically and during inammatory processes. (B) BloodCSF
barriers. The cellular, protein and electrolyte composition of the CSF strikingly differs from that of the blood, reecting the existence of a bloodCSF barrier. (B1) The
ventricular bloodCSF barrier. The function of the choroids plexus is to secrete the CSF in the brain ventricles. In contrast to capillaries of the CNS, blood capillaries of the
choroids plexus are fenestrated. Ependymal secretory cells that actively secrete the CSF into the ventricular lumen are anchored by junctional complexes (apical tight
junctions and basolateral adherens junctions). The ependymal secretory cells are responsible for the ventricular bloodCSF barrier of the choroids plexus. (B2) The meningeal
bloodCSF barrier. When CNS veins exit the parenchyma, they circulate free within the subpial space and brain ssura. When brain veins enter the subarachnoid space, they
are ensheathed by arachnoid cells (green color) that are linked by adherens junctions and are continuous with the pia mater. The venous draining network of the CNS is
therefore a relatively vulnerable part of the bloodCNS barrier.

brain inammation. In vitro cell culture models using human
brain-derived endothelial cell lines that express tight junctions
have played a crucial role in determining how meningitic bacteria
specically interact with the endothelial bloodCNS interface
(Arthur et al., 1987; Weksler et al., 2005). They have permitted the
identication of bacterial adhesins and specic host ligands such
as the platelet activating factor receptor for S. pneumoniae (Cundell
et al., 1995, 1996). However, the precise site and mechanism of
entry of extracellular bacterial pathogens into the CSF is still
enigmatic.
As shown from post-mortem examination of patients who died
of cerebrospinal meningitis (Mairey et al., 2006), extracellular
bacterial pathogens can interact directly with the components of
the bloodbrain barrier and do not need a Trojan horse such as
leukocytes to cross the BBB. It has been thought that bacteria may
preferentially use the choroids plexus (CPs) route to cross the
bloodCSF barrier. However, in this case, meningitis should
theoretically be associated with ventriculitis, which is not
corroborated by clinical and experimental data. Because of their
proximity to the subarachnoidal space and their leaky inter-
endothelial structure, the brain post-capillary venules and veins of
the subpial and subarachnoid spaces may be the site of passage of
bacteria into the CSF. Indeed, once bacteria have crossed the
endothelial monolayer of these vessels, they are separated from
the CSF by only a thin monolayer of leptomeningeal cells. Post-
capillary venules of the parenchyma may also be involved since the
134 O. Join-Lambert et al. / Progress in Neurobiology 91 (2010) 130139

VirchowRobin perivascular spaces are continuous with the
subpial space.
3.2. How do extracellular bacteria break the bloodCSF barrier?
There are at least four possible strategies for a microorganism to
cross a monolayer of endothelial cells (Fig. 3A and B):
(i) transcellular transport by passive or adhesion-induced trans-
cytosis,
(ii) paracellular passage through opened tight junctions,
(iii) disruption of the endothelial barrier due to a direct cytotoxic
effect,
(iv) leukocyte-facilitated transport by infected phagocytes.
These routes are not exclusive, as shown for virus entry into the
CNS that may both directly interact with the bloodbrain barrier or
be transported by infected leukocytes (Huang and Jong, 2001).
However and as already mentioned, extracellular pathogens
probably do not use leukocytes as vehicles to cross the bloodCSF
barrier. A breakdown of the bloodCNS barrier due to apoptosis or
bacterial cytotoxity is unlikely since lesions such as hemorrhage in
the subarachnoidal space are uncommon during bacterial meningi-
tis. Therefore, the entry of bloodborne pathogens most probably
respects the architecture of the bloodCNS barrier (Koedel et al.,
2002). In this view, adhesion of bacteria to endothelial cells could
induce an intracellular signaling leading to disruption of intercellu-
lar tight junctions or, alternatively, bacteria may induce their own
transcytosis through the cell monolayer. In vitro, transcytosis of
bacteria through human brain endothelial cells has been demon-
strated for S. pneumoniae (Ring et al., 1998), E. coli (Prasadarao et al.,
1999; Stins et al., 1999, 2001), S. agalactiae (Nizet et al., 1997), and
through human umbilical vein endothelial cells for H. inuenzae
(Virji et al., 1991, 1992). Although N. meningitidis can readily be
internalized in vitro within vacuoles in human brain microvas-
cular endothelial cells (Nikulin et al., 2006), the route used by
the meningococcus to cross the endothelial cells has been long
debated (Nassif et al., 2002).
3.3. Role of cytokines and innate immunity in bacterial entry into the
CNS
A high grade and/or a sustained bacteremia is required to induce
bacterial meningitis by extracellular pathogens (Dietzman et al.,
1974; Moxon and Ostrow, 1977; Smith, 1987; Tuomanen, 1996).
Although a high bacteremia probably enhances the opportunity for a
pathogen to adhere and to cross the bloodCNS barrier, this
observation may also suggest that activation of the bloodCNS
interface by circulating bacterial compounds or cytokines released
in response to the systemic infection may promote bacterial entry
into the CSF (Fig. 3C). An emerging concept is that the innate
immune response induced during bacteraemia, although not leading
to septic shock, may increase the permeability of the bloodCNS
barrier and may thus favor bacterial crossing of the bloodbrain
barrier. Various bacterial components can rapidly activate the
cellular components of the innate immune system after their entry
into the body. Experimental models of hematogenous meningitis
and bacteremia have shown that in spite of the diversity of the
signaling pathways induced by meningitic bacteria, the early
inammatory response is somewhat similar (Johansson et al.,
2005; Mogensen et al., 2006; Nassif et al., 1992), particularly
including tumour necrosis factor alpha (TNFa), interleukin-1b (IL1-
b) and IL6 that are also key mediators of septic shock (Thijs and Hack,
1995). Interestingly, the peak of early proinammatory cytokines
experimentally corresponds to the peak of bacteremia and to the
onset of bacterial invasion of the meningeal spaces.

Fig. 3. Putative mechanisms of bacterial entry into the CSF. Several routes can be used by bacteria to cross the endothelial bloodCNS barrier. (A) Since extracellular bacteria
such as N. meningitidis are able to survive and multiplicate free in the serum, the rst requisite for free circulating bacteria (in green) to enter into the CSF is direct adhesion to
the cellular layer that forms the front line of the bloodCSF barrier. Translocation through the endothelial cells can occur via the transcellular route, a pathway mediated by
adhesion-induced transcytosis. This mechanism of endothelial BBB crossing is suspected for H. inuenzae, S. pneumoniae, E. coli and S. agalactiae. Bacterial crossing of the
endothelial barrier could also occur through the interendothelial paracellular space, a route that requires the opening of tight junctions. Finally, a direct cytotoxic effect or
bacterial induced apoptosis may also allow free bacteria to enter into the perivascular space. However, this mechanism is unlikely since hemorrhage in the subarachnoidal
space is uncommon during bacterial meningitis. (B) In contrast to intracellular pathogens such as L. monocytogenes, that can invade mononuclear phagocyte and survive
within their cytoplasm, extracellular bacteria resist phagocytosis. Therefore, extracellular bacterial crossing of the bloodCNS barrier is unlikely to occur via circulating
infected phagocytes. (C) The activation of innate immunity that is triggered by invading pathogens at the onset of systemic infections could favor bacterial adhesion and
crossing of the bloodCSF barrier by increasing the expression of membrane ligands to bacterial adhesins on endothelial cells, or by increasing the permeability of the cellular
monolayer. Activation of endothelial cells can be induced either by bacterial circulating molecules, or via the synthesis of proinammatory cytokines released in the blood or
produced by perivascular cells such as astrocytes, perivascular macrophages or microglial cells.
 O. Join-Lambert et al. / Progress in Neurobiology 91 (2010) 130139 135

The role of proinammatory cytokines in the entry of
microorganisms into the CNS has been recently demonstrated
with an experimental model of West Nile virus encephalitis
(Wang et al., 2004). In this study, the authors showed that,
although toll-like receptor 3 (TLR3, a ligand of double-stranded
RNA) was required to control systemic infection, TLR3 decient
mice were more resistant to lethal West Nile virus infection and
showed lower neuropathology in comparison with wild type
mice. TLR3 signaling particularly induced the synthesis of TNFa,
IL1 and interferon b, and the peak of TNFa corresponded to the
time of entry of the virus into the CNS. Focusing on TNFa
signaling, the authors demonstrated that TNFa receptor 1 was
involved in the disruption of the bloodbrain barrier upon TLR3
stimulation by double-stranded RNA. In vitro, TNFa signaling
has also been implicated in the entry of HIV-1 both in the brain
endothelial and trophoblastic cells (Fiala et al., 1997; Parry et al.,
2006).
4. The example of cerebrospinal meningitis
N. meningitidis is the etiological agent of cerebrospinal
meningitis, which occurs mostly in children below 1 year of age
and in young adults. This disease is still responsible for over 30,000
deaths worldwide annually (Fig. 4). Paradoxically N. meningitidis is
a frequent asymptomatic colonizer of the human nasopharynx,
and only a very small proportion of infections proceed to a
sustained bacteraemia and thence to meningitis or septicemia. The
reasons why disease occurs in some individuals and not in others
remain unclear, but human genetic polymorphism is likely to be
important in determining the outcome of infection. The most
important factors predisposing individuals to disease are the
absence of bactericidal antibodies and dysfunction in the
complement system. Some polymorphism of the TLR4 receptor
has also been associated with meningococcal disease. In addition,
all meningococci do not have the same pathogenic potential.
Indeed, analysis of results from multilocus sequence typing (MLST)
has demonstrated the existence of distinct phylogenetic groups
(clonal complexes), some of which are more likely to be isolated
from patients than others. These are the so-called hyper-virulent
or hyperinvasive lineages. Recently, the presence of a prophage
has been shown to be responsible for a large proportion of
invasiveness of strains belonging to hyperinvasive lineages (Bille et
al., 2005). In addition, there is a clear association between the
presence of this prophage and virulence in young adults but not in
children less than 2 years of age (Bille et al., 2008). This element
inserted into the chromosome can be induced to produce a
lamentous phage. Experiments attempting to identify the
phenotype encoded by the phage that is responsible for
hyperinvasiveness have not been conclusive. Indeed deletion of
the entire prophage did not have any effect in laboratory models of
meningococcal pathogenesis. Several observations suggest that
this prophage is associated with genomic rearrangements. These
modications could be associated with an increased tness of the
bacteria for bloodstream dissemination.
Pathology is initiated when the bacteria reach the bloodstream.
Very surprisingly in some patients the bacteria are cleared from the
bloodstream and the only symptom is a febrile u-like syndrome.
Absence of bacterial clearance from the bloodstream paves the way
to fulminant septicemia and/or crossing of the bloodCSF barrier.
Fulminant septicemia has a mortality rate varying between 20%
and 30%. This disease is characterized by a rapidly evolving septic
shock and disseminated intravascular coagulation. Skin hemor-
rhages (purpura fulminans) and a high number of replicating
bacteria in the organs are the hallmark of this form of disease. Even
though bacteria may cross the bloodbrain barrier during the
course of a fulminant septicemia, inammation does not have the
time to occur. The second classical event following bloodstream
invasion by N. meningitidis is the crossing of the bloodCSF barrier
and the initiation of a non-specic inammation in the subarach-
noidal space. As already mentioned, an estimated 63% of
bacteraemia due to N. meningitidis are associated with meningitis,
whereas only 9% of pneumococcal bacteremia is associated with
meningeal invasion (InVS, 2009). Such a high specicity is probably
a consequence of the bacterial adaptation to growth conditions in

Fig. 4. Worldwide epidemiology of meningoccal disease. Meningococcal disease can be sporadic or associated with small outbreaks or large epidemics (Harrison et al., 2009).
Large epidemics only occur in the meningitic belt of sub-Saharan Africa with an estimated incidence that can be as high as 1000 cases per 100,000 population. Although N.
meningitidis serogroup A has been historically and recently responsible for most epidemic outbreaks in Africa, an emergence of N. meningitidis serogroups C, X and W-135 has
been recently observed, causing regional outbreaks. In other regions of the world, the baseline incidence of meningococcal disease usually ranges from 0.5 to 3 per 100,000
population. In Europe, America and Australia, serogroups B and C predominate whereas serogroups A and C are found in Asia. During the last 20 years, N. meningitidis
serogroup Y has emerged in North-America. Recent epidemics:. Saudi Arabia (Hajj pilgrimage), 20002001, serogroup W-135 clone.. Brasil, 1990s (incidence of 6/100,000),
serogroup B and C isolates.. New Zealand, 1990s, serogroup B clone (peak of incidence of 17.4/100,000 in 2001).. United Kingdom, late 1990s, serogroup C clone.. France,
Normandy, since 2003, serogroup B clone.
136 O. Join-Lambert et al. / Progress in Neurobiology 91 (2010) 130139
the human serum, and of the tight interactions of the bacteria with
capillary endothelial cells.
As for neonatal meningitis, the level of bacteremia is directly
correlated with meningeal invasion by N. meningitidis. The
bacterial attributes involved in the growth and/or the survival in
the extracellular uids are therefore playing an essential role in
meningeal invasion by N. meningitidis. Some of these virulence
factors are commonly observed in most extracellular pathogens.
They participate in the prevention of bacterial killing by the host
effectors of the innate immune systems such as polymorphonu-
clear neutrophils and the complement. These bacterial attri-
butes are the polysaccharidic capsule, the lipooligosaccharide
(LOS), and the iron chelation systems. A new virulence factor,
the factor-H binding protein (fHBP), was recently identied
(Seib et al., 2008). It is a 28 kDa surface-exposed lipoprotein that
binds factor-H, a key inhibitor of the complement alternative
pathway. This protein is expressed by all N. meningitidis strains
studied to date, although the level of expression varies between
strains (high, intermediate, or low expressers). Antibodies
directed against fHBP are bactericidal and this protein is
currently one of the best vaccine candidates (Beernink et al.,
2006). The bacteremia is believed to favor meningeal invasion
by directly increasing the likelihood of the interaction of the
bacteria with the components of the bloodCSF barrier;
however, it should be pointed out that the role of the innate
immune effectors in the opening of the bloodCSF barrier
remains unknown.
Very few septicemic extracellular bacterial pathogens have
the ability to adhere and interact as tightly with endothelial
cells as N. meningitidis. Indeed, post-mortem examination of a
case of fulminant meningococcemia clearly demonstrated that
N. meningitidis interacts freely as an extracellular pathogen with
the endothelial cells of the skin, kidney, spleen, liver and brain
(Guarner et al., 2004; Mairey et al., 2006). The interaction of N.
meningitidis with endothelial cells that form the front line of the
bloodCSF barrier plays an important role in the invasion of the
meningeal spaces. In vitro, adhesion of N. meningitidis to
endothelial cells is restricted to human cells, another clue of
the high degree of species specicity of the meningococcus that
is a pathogen restricted to human. More generally, the extensive
adhesion of N. meningitidis to the vessel walls throughout the
body could be responsible for the loss of integrity of the vessels
seen in fulminant meningococcemia and for the extravasation of
red blood cells responsible for the petechies. Finally, in vitro and
ex vivo studies have also shown that N. meningitidis adheres to
human meninges and meningeal cells (Hardy et al., 2000), a
process that is probably critical for N. meningitidis to cross the
bloodCSF barrier and disseminate through the meningeal
spaces.
In vivo, blood ow generates mechanical forces that vary
depending on the vessels and that could prevent bacterial
interaction with the endothelial cells. The ability of N. meningitidis
to bind to endothelial cells in the presence of shear stress
mimicking the bloodstream was recently investigated (Mairey et
al., 2006). These data revealed that, after initial attachment,
bacteria resist high blood velocities, multiply, and form micro-
colonies. This resistance to shear stress and the ability to grow at
the luminal surface of endothelial cells in the presence of blood
ow highlight the efcacy of the interaction between N.
meningitidis and the host cells, for which N. meningitidis has
evolved efcient attributes. Bacterial adhesion involves signaling
to the endothelial monolayer, nally allowing the crossing of the
barrier and meningeal invasion. Both adhesion and signaling
events are promoted by the type IV pili. Ex vivo, type IV pili have
been shown to mediate adhesion not only to human endothelial
cells but also to the meninges (Hardy et al., 2000).
4.1. Type IV pili are the major meningococcal attributes allowing
blood-carried bacteria to interact with CNS endothelial cells
Various bacterial surface components have been described as
allowing the interaction of N. meningitidis with human cells. These
are type IV pili (Tfp) and other attributes such as Opa or Opc proteins
and more recently the secreted component of a two-partner
secretion system. However, in capsulated bacteria, only type IV pili
promote bacterial adhesion since non-piliated capsulated bacteria
are unable to adhere to any cell types. Early work performed with
piliated capsulated meningococci has shown that meningococcal
interaction with human cells can be divided into two steps. The rst
one allows the adhesion of single diplococci in a rather inefcient
manner. The second step corresponds to the bacterial division onto
the apical surface of the cells. Therefore the high number of bacteria
that interact with cells is a consequence of bacterial division of the
few meningococci that have initially succeeded to adhere. The type
IV pili are required for these two steps. They promote the initial
interaction of diplococci with the endothelial cells, and then they
generate bacteria-bacteria interactions that lead to the spreading of
the bacteria on the cell surface.
Type IV pili are polymeric laments found on many Gram-
negative bacteria. These structures correspond to the multimeric
assembly of a pilin subunit protein encoded by the pilE gene. The
pilin subunit (PilE) is synthesized as a preprotein and is produced
by cleavage of a N-terminal sequence of prepilin by the prepilin
peptidase PilD (Lory and Strom, 1997). Functional Tfp are dynamic
structures. Pilin subunits are constantly being assembled into
bers from a platform in the inner-membrane. The ber is then
extruded through the outer membrane. Retraction of the surface-
exposed ber is a key-specicity of Tfp. Retraction is a consequence
of the disassembly of pilin subunits that are then stored in the
cytoplasmic membrane.
4.2. Signaling triggered by N. meningitidis that leads to the
extravasation of brain vessels
4.2.1. Tight adhesion and microcolony formation at the endothelium
surface
After initial attachment to endothelial cells, encapsulated
meningococci proliferate and form microcolonies. The formation
of these colonies is due to Tfp-driven bacteria-bacteria interactions
that allow their spreading at the cell surface, a locomotion mode
reported as twitching motility. When adhering to the apical
membrane of epithelial cells, N. meningitidis induces the local
elongation of microvilli towards the bacteria (Fig. 5A), leading to
the engulfment and internalization of a small proportion of
adhering bacteria (Merz et al., 1996; Pujol et al., 1997).
Interestingly, the formation of such endothelial cell protrusions
was also observed ex vivo by transmission electron microscopy
analysis of brain sections from a child who died from fulminant
meningitis (Pujol et al., 1997). In vitro, adhesion of N. meningitidis to
endothelial cells promotes the formation of membrane protrusions
that surround bacteria, reminiscent of epithelial microvilli
structures, which initiate their internalization within intracellular
vacuoles (Eugene et al., 2002). Surface endothelial cell projections
promotes a tight adhesion of the bacterial colony that is probably
required in vivo to stand up to the shear stress of the bloodstream
(Mairey et al., 2006). These observations strongly suggest that such
morphological modications of the host cell membrane may be
essential for N. meningitidis to cross human vascular endothelium.
4.2.2. Adherent bacteria induce an intracellular signaling that leads to
a localized cytoskeleton remodeling of endothelial cells
The formation of endothelial cell membrane protrusions by
encapsulated N. meningitidis stems from the organization of
 O. Join-Lambert et al. / Progress in Neurobiology 91 (2010) 130139 137

Fig. 5. Signaling pathways triggered by N. meningitidis leading to the formation of the cortical plaque and re-routing of junctional adhesion molecules. (A) Type IV pili
mediated adhesion of N. meningitidis to endothelial cells leads to the formation of membrane protrusions that results from the organization of specic cytoplasmic molecular
complexes, referred as cortical plaques. (1) Endothelial cell protrusions are enriched in ezrin, a members of the ERM (ezrinradixinmoesin) protein family, that not only
controls the organization of the cortical cytoskeleton and leads to cortical plaque formation but also binds the cytoplasmic domain of several transmembrane proteins such as
CD44 or ICAM-1, -2 and E-selectin that cluster at the meningococcus adhesion site. (2) Cortical actin requires the recruitment and phosphorylation of cytoplasmic cortactin.
Two signaling pathways leading to cortactin recruitment beneath bacterial colonies have been identied: the activation of Cdc42 GTPases that recruit the Par6/aPKC polarity
complex and allows the recruitment of the p120-catenin/Cortactin/Arp2/3 complex beneath bacterial colonies, and the activation of the PI3-Kinase/Rac1 GTPase signaling
pathway. Cortactin activation by phosphorylation is required for the formation of actin-rich cell projections. N. meningitidis induces phosphorylation of cortactin by clustering
and activating the host cell tyrosine kinase receptor ErbB2 that activates downstream the src kinase. (3) Cdc42 mediated activation of Par3/Par6/aPKC elicits the recruitment
of adherens and tight junction proteins from pre-existing junctional complexes. (B) Infection of human endothelial cells by Neisseria meningitidis induces the formation of
cortical plaques beneath bacterial colonies. Immunouorescence and confocal microscopy of human endothelial cells infected for 3 h with N. meningitidis (bacteria are
colored in blue with DAPI, whereas ezrin and actin are colored in green and red respectively).

specic cytoplasmic molecular complexes, referred as cortical
plaques, beneath bacterial colonies (Fig. 5A and B). These
protrusions are enriched in ezrin and moesin, two members of
the ERM (ezrinradixinmoesin) protein family that control the
organization of the cortical cytoskeleton through their carboxy-
terminal F-actin binding sites. ERM proteins also bind the
cytoplasmic domain of several ERM binding transmembrane
proteins such as CD44 or ICAM-1, -2 and -3 through their
carboxy-terminal domain. In vitro, it has been demonstrated that
the formation of endothelial cell membrane protrusions by N.
meningitidis are associated with a signaling that leads both to
cortical plaque formation and to the clustering of CD44 or ICAM-1
through the recruitment of the ERM proteins that act as linkers
between the actin cytoskeleton and the plasma membrane
(Eugene et al., 2002; Merz et al., 1999; Merz and So, 1997)
(Fig. 5A and B).
It has been shown that N. meningitidis-induced cortical actin
polymerization relies on the activation of RhoA and Cdc42 GTPases,
and on the activation of a PI3-K/Rac1 GTPase signaling pathway
that is involved in cortactin recruitment at the bacterial adhesion
site (Fig. 5A) (Eugene et al., 2002; Lambotin et al., 2005). Cortactin
(cortical actin binding protein) is a perinuclear cytoplasmic protein
that is involved in the reorganization of the cortical actin
cytoskeleton. Both cortactin recruitment at the N. meningitidis
cell membrane adhesion site and cortactin activation by phos-
phorylation are required for the formation of the actin-rich cell
projections, since inhibition of cortactin translocation or phos-
phorylation leads to structurally altered cortical actin polymeri-
zation (Lambotin et al., 2005). Interestingly, endothelial N.
meningitidis-induced phosphorylation of cortactin results from
the clustering and activation of the host cell tyrosine kinase
receptor ErbB2 that activates downstream the kinase src (Fig. 5A)
(Hoffmann et al., 2001). ErbB2 belongs to the family of epidermal
growth factor (EGF) receptors, and the interaction of N. meningitidis
with human endothelial cells leads to the activation of ErbB2, most
likely via formation of ErbB2 homodimers.
The above signaling is observed only with piliated bacteria,
thus demonstrating that Tfp are required for these rearrange-
ments to occur. Furthermore, this signaling is greatly enhanced
when mechanical force is exerted on the host cell membrane
(Merz and So, 2000). It is interesting to note that the signaling
pathways promoted by N. meningitidis, summarized in Fig. 5A,
are similar to those induced by leukocyte adhesion on
endothelial cells (Doulet et al., 2006). Leukocyte adhesion
promotes the remodeling of the apical endothelial plasma
membrane into projections that surround adherent leukocytes
(Barreiro et al., 2005; Carman and Springer, 2004; Shaw et al.,
2004). These structures, referred to as endothelial docking
structures or transmigratory cups, are essential to promote
rm adhesion and extravasation of leukocytes through para-
cellular as well as transcellular routes. Transmigratory cups
result from the dynamic redistribution of ICAM-1, VCAM-1 and
CD44 at the endothelialleukocyte contact area, accompanied by
the recruitment of activated ERM proteins, and lead to cortical
actin polymerization. Therefore, the same set of endothelial
proteins is present in the membrane protrusions induced by N.
meningitidis and in the docking structures promoted by
leukocyte adhesion. In addition, N. meningitidis colonies on
the endothelial surface prevent the accumulation of leukocytes
and the formation of docking structures by inducing the massive
recruitment of ezrin and moesin together with ICAM-1, ICAM-2,
VCAM-1, and CD44 (Doulet et al., 2006). These data strongly
138 O. Join-Lambert et al. / Progress in Neurobiology 91 (2010) 130139
suggest that, when adhering to endothelial cells, N. meningitidis
hijacks the cellular machinery normally used for leukocyte
trans-endothelial migration to promote the formation of the
cortical plaque.
4.2.3. N. meningitidis opens a paracellular route to cross the
endothelial bloodCSF barrier
Although N. meningitidis can readily be internalized within
vacuoles in human brain microvascular endothelial cells (Nikulin
et al., 2006), the route used by the meningococcus to cross the
endothelial cell has been long debated (Nassif et al., 2002). As
mentioned above for other bacterial pathogen that can cross the
bloodCSF barrier, an appealing hypothesis was transcytosis. In
vitro data showing that N. meningitidis can cross a monolayer of
epithelial cells with tight junctions without destroying the
intercellular junctions tted well with this hypothesis. However,
this mechanism of bacterial translocation has never been
demonstrated with endothelial cells, and in vitro, only a small
proportion of bacteria are internalized after adhesion to endothe-
lial cells. A recent study has unraveled the mechanisms allowing N.
meningitidis to cross endothelial cells of the CNS vessels (Coureuil
et al., 2009). It has been demonstrated that the polarity complex
Par3/Par6/aPKC that plays a pivotal role in the formation of
intercellular junctions is recruited in the cortical plaque at the site
of the bacterial adhesion. The recruitment of the polarity complex
is sufcient to organize an ectopic intercellular junction-like
domain at the site of bacterial cell interaction. These ectopic
junctions result from the re-routing of cell-cell adherens junctions
such as VE-cadherin, p120-catenin and b-catenin, and of tight
junction proteins such as, ZO-1, ZO-2, and claudin-5. The
recruitment of junctional molecules beneath N. meningitidis
microcolonies is associated with the formation of intercellular
leakages. The relocalisation of these junctional proteins at the site
of bacterial cell adhesion opens the BBB, thus allowing the bacteria
to cross the endothelial monolayer (Coureuil et al., 2009).
5. Conclusion
Recent exciting ndings have considerably expanded our
understanding of the cellular events involved in N. meningitidis
invasion processes, underlining the complex sequence of signaling
events induced by N. meningitidis to elicit its uptake in non
phagocytic cells. However, in spite of recent advances in our
understanding of these molecular mechanisms, much remains to
be discovered about the complex molecular networks involved.
Among major issues is the identication of the receptor for
meningococcal pili, which would constitute a signicant break-
through in the eld and pave the way to future developments of
novel vaccine strategies. Considering that pathogenesis is not the
usual outcome of the colonization of a host by N. meningitidis, but
rather an accident, one remaining question is how cellular invasion
helps this bacterium to disseminate. It is likely that invading host
cells at the site of colonization help N. meningitidis to persist inside
its host and to evade mucosal innate immunity.
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