Strategies used by bacterial pathogens to cross the blood–brain barrier

M I C R O R E V I E W

Strategies used by bacterial pathogens to cross the blood

–brain

barrier

Loic Le Guennec

|

_{Mathieu Coureuil}

|

_{Xavier Nassif}

|

Sandrine Bourdoulous

1

Inserm (Institut National de la Sante et de la Recherche Medicale), U1016, Institut Cochin, Paris, France

2

CNRS (Centre National de la recherche Scientifique), UMR8104, Paris, France

3

Université Paris Descartes, Sorbonne Paris Cité, Paris, France

4

Inserm (Institut National de la Sante et de la Recherche Medicale), unité U1151, Institut‐ Necker‐Enfants‐Malades, Paris, France

5

CNRS (Centre National de la recherche Scientifique), UMR 8253, Paris, France

6

Université Paris Descartes, Sorbonne Paris Cité, Faculté de médecine, Paris, France

7

Assistance Publique– Hôpitaux de Paris, Hôpital Necker Enfants Malades, Paris, France Correspondence

Sandrine Bourdoulous, INSTITUT COCHIN, 22 rue méchain, 75014 Paris, France.

Email: sandrine.bourdoulous@inserm.fr Funding information

Fondation pour la Recherche Médicale, Grant/ Award Number: Doctoral fellowship; Université Paris Descartes; CNRS; INSERM; Agence Nationale de la Recherche, Grant/ Award Numbers: ANR‐15‐CE15‐0002, ANR‐ 14‐IFEC14‐0006 and ANR‐14‐CE14‐0010‐01

Abstract

The skull, spine, meninges, and cellular barriers at the blood

–brain and the blood–

cerebrospinal fluid interfaces well protect the brain and meningeal spaces against

microbial invasion. However, once in the bloodstream, a range of pathogenic bacteria

is able to reach the brain and cause meningitis. Despite advances in antibacterial

ther-apy, bacterial meningitis remains one of the most important infectious diseases

worldwide. The most common causative bacteria in children and adults are

Strepto-coccus pneumoniae and Neisseria meningitidis associated with high morbidity and

mor-tality, while among neonates, most cases of bacterial meningitis are due to group B

Streptococcus and Escherichia coli. Here we summarise our current knowledge on

the strategies used by these bacterial pathogens to survive in the bloodstream, to

col-onise the brain vasculature and to cross the blood

–brain barrier.

K E Y W O R D S

bacterial invasion, blood–brain barrier, endothelial cells, inflammation, meningitis, virulence factors

1

I N T R O D U C T I O N

The central nervous system (CNS) is protected by three protective membranes: the skull, the spine, and the meninges. The blood–brain barrier (BBB) is part of the meninges and constitutes a selective filter for a tightly regulated exchange between the blood and the CNS of

water, solutes, nutrients, or compounds. Moreover, it gives a protec-tion against pathogen invasion (Weiss, Miller, Cazaubon, & Couraud, 2009). BBB consists of vessels formed by continuous nonfenestrated specialised endothelial cells expressing selective intercellular tight junction proteins, which restrict the paracellular passage of molecules and of supporting cells as pericytes and smooth muscle cells. The

Abbreviations: ACP, Alpha C protein;β2AR, Beta‐2‐adrenergic receptor; BBB, Blood‐Brain Barrier; CNS, Central Nervous System; CSF, Cerebro‐Spinal Fluid; E. coli, Escherichia coli; EoD, Early‐ onset Disease; fHBP, factor H‐binding protein; GBS, Group B Streptococcus; H. influenzae, Haemophilus influenzae; HvgA, Hypervirulent GBS adhesin; IL, Interleukin; L. monocytogenes, Listeria monocytogenes; LoD, Late‐onset Disease; LTA, Lipoteichoic acid; N. meningitidis, Neisseria meningitidis; MMP, Matrix metalloproteinase; OmpA, Outer membrane protein A; PAF, Platelet‐ activating factor; Pcho:, Phosphorylcholine; pIgR, Polymeric immunoglobulin receptor; PMN, Polymorphonuclear neutrophil; ROS, Reactive oxygen species; RNS, Reactive nitrogen species; SCID, Severe Combined ImmunoDeficient; Sfb, Streptococcal fibronectin‐binding; S. pneumoniae, Streptococcus pneumoniae; Srr, Serine‐rich repeat; T4P, Type IV pili; TNF‐ α, Tumor necrosis factor‐alpha; TRL, Toll‐like‐receptor; ZO1, Zona Occludens 1

DOI: 10.1111/cmi.13132

Cellular Microbiology. 2020;22:e13132. https://doi.org/10.1111/cmi.13132

© 2019 John Wiley & Sons Ltd

vascular basement membrane, in close contact with the astrocytic foot processes, also displays organ_{‐specific architectural characteristics and} forms a limiting membrane around the CNS.

These architectural characteristics limit the traffic of host immune cells (granulocytes, T, and B cells) into the brain parenchyma. Host defence in the CNS is not optimal to control potential bacterial inva-sion and pathogenicity, and mostly relies on innate immune cells including parenchymal (microglia) and nonparenchymal macrophages and perivascular blood‐derived monocytes (Prinz & Priller, 2017). This particularity has been called_{“CNS immune privilege”. The presence of} a lymphatic vasculature within the brain has been recently discussed but is still controversial (Aspelund et al., 2015).

Despite the CNS protection, few bacteria are able to cross the BBB and promote acute meningitis. In children and adults, bacterial menin-gitis is mainly caused by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae and during the neonatal period by Escherichia coli and group B Streptococcus (GBS). One partic-ular case is Listeria monocytogenes, which affects both neonates and immunocompromised, or elderly patients. Among less common bacte-ria responsible for acute meningitis, we can mention nontyphoideal Salmonella, Staphylococcus aureus, Mycobacterium tuberculosis, Klebsi-ella spp., and Streptococcus suis (Coureuil, Lecuyer, Bourdoulous, & Nassif, 2017; Doran et al., 2016; Wilkinson et al., 2017). All these pathogens have the ability to asymptomatically colonise the upper respiratory tract, the skin, and the different mucosal surfaces. They can reach the bloodstream, survive within the blood and finally, cross the BBB. To interact with host cells or escape immune responses, these bacteria have evolved specific or common skills.

Despite advances in antibiotic therapy and vaccines, bacterial men-ingitis is still a worldwide global health issues with high morbidity and mortality. Complications frequently occur, leading to high proportion of patients with unfavourable outcome and to neurological sequelae in survivors (Leber et al., 2016). Rapid diagnosis and prompt treatment are therefore required, and advance in knowledge of mechanisms of bacterial BBB invasion is still mandatory.

In this review, we first describe the common steps in bacterial meningitis. We then detail the most frequent bacteria responsible for acute meningitis and their respective pathophysiological mecha-nisms leading to mucosal colonisation, haematogenous dissemination, and crossing of the BBB, which are summarised in Figure 1.

1.1

Common steps in bacterial meningitis

Pharyngeal, respiratory, or digestive mucosal surface colonisation is often a prerequisite for the pathogenesis of bacteria responsible for meningitis. A hallmark of these bacteria is their capacity to persist in the circulation by resisting polymorphonuclear neutrophil (PMN) phagocytosis and complement killing. Except for N. meningitidis, a sustained and prolonged bacteraemia often correlates with meningeal invasion and is required to provoke meningitis (Brandt et al., 2008; Smith et al., 1982). However, bacteria causing mastoid and sinus infec-tion may also penetrate through local skull lesions or through perforat-ing nutrient vessels that supply the skull. Nevertheless, followperforat-ing

bacteraemia or spreading from bacterial foci contiguous to the brain, all bacteria have to breach the BBB or the blood_{–CSF barrier to} ulti-mately invade the brain.

In recent years, many advances have been made in the understanding of the mechanisms responsible for the interaction of pathogenic bacteria with brain vessels, leading to the identification of key bacterial adhesins and of their cognate endothelial cell receptors. However, as most of these pathogens are human specific, study of their pathophysiology is limited by the lack of experimental animal models mimicking all the features of the human disease. The precise mechanisms by which these pathogens cross through the BBB, as well as their exact anatomical site of entry are still poorly known. It has been suggested that bacteria could translo-cate through the choroid plexus to reach the brain, but in this hypothesis, bacterial meningitis should be associated with prior ventriculitis, a clinical presentation which has been rarely observed. It is more likely that bacte-ria cross the BBB through postcapillary perforating venules because of the low blood flow in these latter, which could facilitate bacterial adhe-sion to brain endothelial cells and because of their proximity with the sub-arachnoid space, the Virchow Robin space, where endothelial cells express less selective intercellular junction proteins and cerebrospinal fluid (CSF) (Coureuil et al., 2017).

Once interacting with brain endothelial cells, bacteria can cross the BBB by different ways: by invading and crossing through brain endothe-lial cells (transcellular pathway), by disrupting intercellular junctions and/or inducing cell damage (paracellularly), or in infected phagocytes (Trojan_{‐horse mechanism). This latter mechanism is more likely to} corre-spond to intracellular bacteria as L. monocytogenes. As most of the bac-teria responsible for meningitis are extracellular bacbac-teria, which have acquired virulence factors allowing phagocytic resistance, it is most unlikely that those might use this route to cross the BBB. Moreover, several studies have shown a direct interaction between these extracel-lular bacteria and brain endothelial cells in vivo (Coureuil et al., 2017; Doran et al., 2016). Upon adhesion to brain endothelial cells, these path-ogens may induce their own internalisation in brain endothelial cells and promote their passage by a transcellular route, or induce intracellular signalling pathways, which may lead to the disruption of the intercellular tight junctions and bacterial crossing of the BBB by a paracellular route. Some bacteria, like S. pneumoniae, GBS, and E. coli, produce cytolytic toxins that alter host cells, leading to barrier disruption, thereby opening the path to bacterial invasion by a paracellular route. Interestingly, even if they are able to cross the BBB and to disrupt intercellular tight junc-tions, these pathogens seem to respect the architecture of the BBB, as subarachnoid haemorrhages are rarely observed during bacterial menin-gitis (Koedel, Scheld, & Pfister, 2002; Lang, Leach, Emelifeonwu, & Bukhari, 2013; Rajendran, Behrens, & Pawley, 2018). Finally, another conceivable pathway, independent of a preceding bacteraemia, to reach the subarachnoid meninges could be through an axonal transport via the olfactory nerve (Dando et al., 2014). This route is limited to pathogens colonising the nasopharyngeal mucosa. It has been described for S. pneumoniae and N. meningitidis in mice (Sjolinder & Jonsson, 2010; van Ginkel et al., 2003).

Inflammation also plays a central role in bacterial crossing of the BBB. In response to bacteraemia, with or without septic shock, and

to CNS infection, endothelial cells, microglial cells, astrocytes, ependymal cells, and resident macrophages secrete proinflammatory molecules (Moreillon & Majcherczyk, 2003). Although it is required

for host protection against bacterial agents, this innate immune response increases BBB permeability and could promote bacterial entry through the meninges. It is also responsible for inflammation‐ FIGURE 1 Mechanisms involved in bacterial crossing of the blood–brain barrier Streptococcus pneumoniae adheres to endothelial cells by using bacterial phosphorylcholine (PCho), which mimics the chemokine platelet_{‐activating factor (PAF) and binds a cell receptor called PAF receptor} (PAFr); the choline‐binding protein CbpA, which binds to the laminin receptor (LR); the surface neuraminidase A (NanA), which binds to LR and the platelet endothelial cell adhesion molecule_{‐1 (PECAM‐1); and the pilus‐related adhesion RrgA, which bind to PECAM‐1 and to the poly}

immunoglobulin receptor (pIgR). These interactions enable bacterial transcytosis across the endothelium. In addition, bacterial pneumolysin and H2O2

release promote cell wall degradation and consequently endothelial monolayer disruption, allowing bacterial invasion through a paracellular pathway. Neisseria meningitidis interacts with endothelial cell via interaction between their type IV pili and the cellular receptor CD147. Following initial bacterial adhesion, type IV pili bind to the_{β2‐adrenergic receptor promoting signalling events that lead to the formation of membrane protrusions} enabling bacteria to resist the forces exerted by the blood flow and to proliferate at the endothelial cell surface, a process referred to as vascular colonisation. In addition, pilus_{‐mediated signalling events promote alteration in the tight junction organization, opening the path to the bacterial} crossing of the blood–brain barrier through a paracellular pathway. Hypervirulent group B Streptococcus adhesins (HvgA), alpha‐C protein (ACP), Streptoccocal fibronectin_{‐binding protein (SfB), and PilA promote group B Streptococcus interaction with endothelial cells, some through interaction} with extracellular matrix components, such as fibronectin or collagen, which bind to cell surface receptors, or through interaction with

glycosaminoglycans. In addition, the bacterial serin rich repeat protein 2 (Srr2) binds to plasminogen and plasmin, which promote cell wall degradation, and consequently, endothelial monolayer disruption. Moreover, group B Streptococcus expresses a pore formingβ‐haemolysin, which is cytolytic for human brain endothelial cells and may contribute to BBB disruption, allowing bacterial invasion through a paracellular pathway. The Escherichia coli K1 adhesins“outer membrane protein A” (OmPA) and “invasion of the brain endothelium protein A” (IbeA), which bind respectively to endothelial receptors_{“beta‐form of the heat‐shock gp96” (Ecgp96) and contactin‐associated protein 1 (CaspR1), promote bacterial transcytosis across the} endothelium. In addition, the haemolysin‐co‐regulated protein 1 (Hcp1), a component of theType VI secretion system of E. coli K1, can be injected in the cytoplasm of human brain endothelial cells and induces apoptosis, leading to cell necrosis and bacterial crossing via a paracellular pathway

induced adverse events, such as BBB disruption, leukocyte invasion in the CSF, and neuronal injuries. Indeed, cytokines, such as tumour necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, IL‐8 and IL‐6, initiate inflammatory cascade, immune cell attraction, but also matrix metallo-proteinase (MMPs) production, which degrade the extracellular matrix, increasing BBB permeability and allowing cell migration (Sellner & Leib, 2006). This inflammation cascade promotes the expression of adhesion molecules at the endothelial cell surface, such as VCAM_‐1 and ICAM‐1, which mediate adhesion of various leukocyte types (Greenwood, Etienne_{‐Manneville, Adamson, & Couraud, 2002). All} these events favour neutrophil extravasation in the brain. In addition, during acute meningitis, brain endothelial cells produce endothelin, a strong vasoconstrictor, which contributes to cerebral blood flow reduction (Eisenhut, 2014) and granulocytes; endothelial cells and microglial cells produce reactive oxygen species (ROS) and reactive nitrogen species (RNS), which also contribute to brain vascular dam-ages (Schaper et al., 2002). At last, neuronal damdam-ages secondary to inflammation are observed, responsible for long_{‐term neurological} sequelae. Nuclear factor kappa B (NF‐kB) pathway activation is heavily implied in this neuronal degeneration (Amor, Puentes, Baker, & van der Valk, 2010; Lawrence, 2009). Diosmetin, an inhibitor of the heme containing enzymes CYP1B1 and CYP1A1 that participate in the metabolism of various xenobiotics and endogenous substances (Androutsopoulos et al., 2013), can also antagonise the activation of NF‐kB signalling pathway and reduce inflammation, neuronal apopto-sis, and neurological disabilities in a rat model of S. pneumoniae menin-gitis (Liu et al., 2019). In clinics, leukocyte invasion secondary to bacterial BBB crossing is generally prevented by adjuvant corticoste-roid therapy, which improves mortality and reduces neurological sequelae, including deafness (de Gans & van de Beek, 2002).

1.1.1

Streptococcus pneumoniae

S. pneumoniae (pneumococcus) is a Gram_{‐positive extracellular coccus.} It resides in the upper respiratory tract, sinuses, and nasal cavity as an asymptomatic coloniser and can be responsible for otitis, sinusitis, and severe invasive infection, such as pneumonia, bacteraemia, and acute meningitis. Neurological complications secondary to pneumococcal meningitis, such as cerebral swelling, brain infarction, and intracranial hypertension, are common and contribute to the high mortality rate (20%–30%) and to disability among the survivors. In order to reach brain endothelial cells, pneumococci have to escape ear_{‐nose‐throat} or respiratory mucosal defences to cross epithelium of the nasopha-ryngeal barrier and to survive in the bloodstream.

Our understanding of pneumococcus physiopathology arises from the study of brain autopsies and from animal models (mouse, rabbit, and rat) that closely mimic clinical features of human disease. High titer bacteraemia is mandatory to promote meningitis (Iovino, Seinen, Henriques‐Normark, & van Dijl, 2016; Iovino, Sender, & Henriques‐ Normark, 2019). The polysaccharide capsule provides protection to S. pneumoniae by interfering with phagocytosis, complement‐mediated immunity, and opsonophagocytic killing and by masking several Toll_‐ like‐receptor ligands within the bacterial cell wall, including

lipoteichoic acid and lipopeptides, therefore preventing the triggering of innate immune responses. Numerous S. pneumoniae meningitis_‐ associated factors have been reported that target host cell receptors degrade the extracellular matrix or cell intercellular junctional compo-nents. The first described was the pneumococcal phosphorylcholine (PCho), which binds to the platelet_{‐activating factor receptor (PAFr)} by a molecular mimicry mechanism with the chemokine PAF. This interaction could promote bacterial translocation across the BBB (Ring, Weiser, & Tuomanen, 1998). Afterwards, several pneumococcal surface proteins were described to promote adhesion to host cell receptors. Among those is a choline‐binding protein called CbpA (also called PspC, SpsA or Hic), which binds the laminin receptor (LR) (Orihuela et al., 2009). Interestingly, N. meningitidis and H. influenzae can both use a choline_{‐binding protein, which shares homology with} CbpA, to bind LR and to mediate bacterial adhesion to brain endothe-lial cells (Orihuela et al., 2009). This observation led to the production of vaccine based on CbpA protein that cross‐protects against these bacteria (Mann et al., 2014). The surface neuraminidase A (NanA), which interacts with both LR and the platelet endothelial cell adhesion molecule_{‐1 (PECAM‐1), can also contribute to pneumococcal} attach-ment to the BBB (Uchiyama et al., 2009). The pneumococcal pilus‐1 and the pilus_{‐related adhesin RrgA, which increase attachment to} brain endothelial cells, also facilitate BBB translocation through bind-ing to poly immunoglobin receptor (pIgR) and PECAM_{‐1 (Iovino} et al., 2017). Finally, the pneumococcus can also use the vitronectin‐ receptor (_{αvβ3 integrin, CD51/CD61) to invade brain endothelial cells} (Bergmann et al., 2009). However, encapsulation, found in most clini-cal isolates, is unfavourable to successful colonisation, as it masks the binding sites of pneumococcal surface proteins that are required to bind to host cells and for transcytosis (Li_{‐Korotky, Lo, & Banks,} 2010). Reduced levels of capsule are then required to promote binding to the host barriers (Shainheit, Mule, & Camilli, 2014). At initial stages of the infection, capsulated S. pneumoniae strains may thus divert the BBB by a different mechanism.

In addition to receptor‐mediated intracellular uptake, S. pneumoniae may gain brain access through a paracellular pathway by disrupting BBB integrity. This process is mediated by pneumococcal autolysis, an important characteristic of S. pneumoniae, responsible for the release of key virulence factors. Among secreted factors is the pneumolysin (Ply), overrepresented in clinically isolated strains, which binds to cholesterol to form pores within the host cell membrane and the α‐glycerophosphate oxidase GlpO, which generates H2O2. Both induce

tissue damages and disruption of BBB integrity, favouring bacterial entry into the CNS (Paton, Andrew, Boulnois, & Mitchell, 1993). More recently, it was shown that Ply induces high expression of CERB‐ binding protein (CBP) in brain endothelial cells in vitro and in vivo after intravenous injection in mice (Chen et al., 2019). This transcriptional cofactor promotes the release of TNF_{‐α and apoptosis, resulting in} increased permeability of the BBB. CBP inhibitors could reduce BBB leakage induced by Ply, making CBP an interesting target for future S. pneumoniae therapy (Chen et al., 2019).

Ply expression also seems to play a key role in the fate of S. pneumoniae within brain endothelial cells during passage across the

BBB (Surve et al., 2018). While a majority of high Ply producing subset of S. pneumoniae mediates damage to their pneumococcus containing vacuolar membrane leading to recruitment of cytosolic“eat‐me” sig-nals and autophagic clearance, low Ply producing S. pneumoniae lead to autophagosomal maturation blockade and evasion of host cell defence mechanisms, therefore extending survival and promoting transcytosis across brain endothelial cells, both in vitro and in vivo (Surve et al., 2018).

Therapeutic strategies, which interfere with S. pneumoniae brain endothelial cell adhesion receptors, have been explored in several in vitro studies in order to develop adjuvant therapies to antibiotics to cure pneumococcal meningitis. For example, chemical antagonists, such as L659989 or WEB2086, known to block PAFR in vitro (Iovino, Brouwer, van de Beek, Molema, & Bijlsma, 2013), or soluble CbpA, which compete witch S. pneumoniae for its binding to endothelial cells, have been tested (Liu et al., 2019; Lu, Lamm, Li, Corthesy, & Zhang, 2003; Zhang et al., 2000). However, the data showed no convincing evidence that these antagonists improved the management of pneu-mococcal meningitis (i.e., lower bacteraemia, increased survival) in mice model studies (Iovino et al., 2013).

To date, there are over 90 distinct capsular types, increasing the challenge of vaccine development. The most widely used vaccines are the 23‐valent pneumococcal polysaccharide vaccine (PPV23) and the 13_{‐valent pneumococcal conjugate vaccine (PCV13), which cover} respectively 23 and 13 serotypes with a good vaccine efficacy to pre-vent invasive pneumococcal infections. A study in Spain showed that, since the PCV13 vaccine was introduced in 2010 in this country, pneumococcal meningitis incidence has been reduced from 2.3/ 100,000 to 0.5/100,000 (Gonzalez‐Escartin, Angulo Lopez, Ots Ruiz, Martinez_{‐Martinez, & Cabero Perez, 2017). However, there is now} an increasing incidence of infections caused by nonvaccine serotypes. Prospects for the future are to develop a whole vaccine, allowing vac-cination against all serogroups (Pichichero, 2017). Safety clinical trials are already under way to evaluate tolerance and effectiveness of S. pneumonia whole cell vaccine recently produced (NCT01537185).

1.1.2

_{Neisseria meningitidis}

N. meningitidis (meningococcus) is a Gram‐negative diplococcus responsible for severe invasive infection, such as acute meningitis and purpura fulminans. Strictly adapted to human, it has an asymptom-atic nasopharyngeal carriage in 10% of the population (Pace & Pollard, 2012). Meningococci are transmitted by saliva and respiratory secre-tions. The prerequisite for meningococcal pathogenicity is the crossing of the mucosal barrier in order to reach the circulation (Coureuil, Bourdoulous, Marullo, & Nassif, 2014). Once in the bloodstream, many virulence factors favour bacterial growth and immune escape from host effectors and complement system (Coureuil et al., 2017; Virji, 2009). Circulating bacteria have the propensity to interact with endo-thelial cells lining microvessels in different locations and to rapidly pro-liferate at the endothelial cell surface to form large bacterial aggregates filling the lumen of blood capillaries (Coureuil et al., 2014). Infection is associated with unregulated activation of

coagulation and inflammation, promoting vascular damage and the appearance of purpuric lesions. As compared to peripheral vessels, the interaction with brain microvessels allows bacteria to cross the BBB with relative conserved integrity of brain endothelial cell junc-tions and no sign of brain haemorrhage or brain vessel thrombosis in necropsies (Pron et al., 1997).

Because N. meningitidis interacts only with human cells, most of the findings regarding the pathophysiology of meningococcal meningitis are derived from in vitro studies on human brain endothelial cells, from ex‐ vivo studies on fresh human brain samples, and from examination of postmortem tissue samples, showing that meningococcal meningitis is associated with the colonisation of brain capillaries in the subarachnoid space, the parenchyma, and the choroid plexus (Mairey et al., 2006; Pron et al., 1997). More recently, a humanised xenografted mouse model has been developed, in which severe combined immunodefi-ciency (SCID) mice were grafted with human skin. As human vessels within the graft connect with the mice vascularisation, they are main-tained functional, allowing in vivo studies. Although it does not provide information on the mechanism of bacterial crossing of the BBB, this model has demonstrated that vascular colonisation of human vessels by meningococci is a prerequisite to events leading to vascular alter-ations, thrombosis, inflammation, the hallmarks of purpuric lesions (Join‐Lambert et al., 2013; Melican, Michea Veloso, Martin, Bruneval, & Dumenil, 2013).

The propensity of encapsulated meningococci to adhere to human endothelial cells relies on type IV pili (Tfp) (Lemichez, Lecuit, Nassif, & Bourdoulous, 2010; Virji, 2009). These long filamentous structures are composed of heteromultimeric pilin subunits, which are assembled into helical fibres by a complex machinery and secreted through a pore in the outer membrane (Craig & Li, 2008). N. meningitidis utilises CD147 for Tfp‐dependent adhesion to endothelial cells (Bernard et al., 2014). CD147 (also called EMMPRIN or Basigin) is a member of the immuno-globulin superfamily highly expressed at the surface of brain capillaries. When incubated with human brain tissue explants ex vivo, meningococci establish specific tight association predominantly with CD147‐positive brain endothelial cells and leptomeningeal cells in Virchow_‐Robin spaces (Bernard et al., 2014). In the cortex, meningococci are found in the vicinity of cortical brain vessels, but they are not associated with glial or neuronal cells that do not express CD147, demonstrating a close correlation between CD147 expression and the propensity of N. meningitidis to selectively colonise brain capillaries and meninges in human brain. Interfering with CD147/Tfp interaction blocks meningo-coccal adhesion to human brain endothelial cells in vitro and prevents human vessel colonisation in brain tissue ex vivo.

Following this initial interaction, Tfp can then interact with the G protein_{‐coupled beta‐2‐adrenergic receptor (β2AR) (Coureuil et al.,} 2010). This interaction does not elicit the canonical G protein‐ mediated signal transduction. However, it results in a biased activation ofβ‐arrestin‐mediated signalling pathways promoting the local recruit-ment and activation of cytoskeleton_{‐associated and signalling} pro-teins. As a result, Tfp‐mediated signalling events induce the local remodelling of the plasma membrane under bacterial colonies, leading to the formation of membrane protrusions that rapidly stabilise

bacterial colonies at the endothelial cell surface, enabling them to resist blood flow_{‐generated shear stress (Mikaty et al., 2009). It was} recently shown that such membrane remodelling is initiated alongside Tfp fibres leading to the formation of complex embedded pili_‐plasma membrane structures that would provide the microcolony with enough mechanical coherence to resist shear stress (Charles_‐Orszag et al., 2018). In addition, Tfp‐pilus mediated signalling events promote the delocalisation of cell_{–cell junction molecules, such as VE‐cadherin,} zonula occludens‐1 (ZO‐1), or claudin‐5, to sites of bacterial adhesion, leading to altered endothelial permeability (Coureuil et al., 2009). Acti-vation of matrix metalloproteinase 8 (MMP8) production can also lead to proteolytic cleavage of the tight junction protein occludin and con-tribute to cell detachment (Schubert‐Unkmeir et al., 2010).

Bacterial binding to endothelial cells also requires reorganisation of the host cell receptors at the plasma membrane. Indeed, although the affinity of Tfp for CD147 and_{β2AR is quite weak, this is compensated} by the assembly of these receptors into highly ordered clusters at bac-terial adhesion sites, increasing the binding strength of meningococci to endothelial cells under shear stress (Maissa et al., 2017). Moreover, specialised raft domains are required for proper assembly of receptors and signalling molecules (Simonis, Hebling, Gulbins, Schneider‐ Schaulies, & Schubert_{‐Unkmeir, 2014). Finally, Tfp‐mediated} colonisa-tion of endothelial cells induces transcripcolonisa-tional regulacolonisa-tion of numerous genes involved in cell survival, inflammation, the expression of adhe-sion molecules, and the secretion of pro‐inflammatory cytokines (Schubert_{‐Unkmeir, Sokolova, Panzner, Eigenthaler, & Frosch, 2007).} These events contribute to both local and systemic inflammation and result in septic shock and multiple organ failure (Sokolova et al., 2004). In addition, a number of other meningococcal minor adhesins, such as surface exposed Opa and Opc proteins (Hardy, Christodoulides, Weller, & Heckels, 2000), the adhesin complex protein (ACP) (Hung, Heckels, & Christodoulides, 2013), the autotransporter meningococcal serine protease A (MspA) (Turner et al., 2006), and potentially others (Kanova et al., 2018), can reinforce and/or modulate adhesion.

Because of the human specificity of this bacterium, there is no rel-evant in vivo experimental animal model yet to assess the molecular mechanism involved in meningococcal meningitis. Whether these events occur in vivo at the BBB remains to be determined.

So far, the best prevention against invasive meningococcal disease is vaccination. Six capsular serogroups (A, B, C, W, X, and Y) are responsible for most cases and form the basis of conjugate vaccines (Rosenstein, Per-kins, Stephens, Popovic, & Hughes, 2001), with the exception of the serogroup B, as the α(2–8) linked sialic acid homopolymer of this serogroup is identical to which of the mammalian neural cell adhesion molecule (NCAM) and is poorly immunogenic (Seifert, Glanz, Glaubitz, Horstkorte, & Bork, 2012). Current vaccine strategies against serogroup B therefore rely on immunisation against noncapsular antigens. However, although these vaccines provide long_{‐term immunological response, their} effect on carriage prevalence is less clear (Balmer, Burman, Serra, & York, 2018). Moreover, in immunocompromised patients, vaccination against serogroup B is unlikely to provide proper protection (Parikh et al., 2017). Currently, trials are ongoing for serogroup B vaccination using ade-novirus as a vector (Morris, Sebastian, Spencer, & Gilbert, 2016).

1.2

_{Group B}

Streptococcus

GBS is a Gram‐positive extracellular diplococcus colonising asymp-tomatically the genitourinary and gastrointestinal tracts of up to 30% of healthy adults. About 30%–70% of neonates born to mothers with GBS colonisation become transiently colonised by their mother's organism, and 1% will develop invasive infection (Edmond et al., 2012). GBS is the first cause of meningitis in neonates. Contamination usually occurs by maternal‐fetal transmission at the time of delivery, by inhalation, or ingestion of amniotic fluid. Bacteria then cross the fetal respiratory or gastrointestinal mucosa, proliferate in the blood, and enter the CNS.

GBS infection in newborns is usually described as being early or late_{‐onset. Early‐onset disease (EoD) typically occurs within the first} week of life, associating pneumonia, bacteraemia and sometimes men-ingitis, whereas late_{‐onset disease (LoD) occurs in infants up to 2} months of age with fewer symptoms related to bacteraemia and a higher incidence of meningitis. Among the ten GBS known serotypes, five are most commonly associated with disease (Ia, Ib, II, III, and V). However, a single clone, GBS ST_{‐17, defines a “highly virulent”} capsu-lar serotype III clone strongly associated with LoD meningitis (Tazi et al., 2012). GBS meningitis accounts for 14% of meningitis mortality. Small amount of PMN and secreted IgA/IgG and absence of alveolar macrophages make neonates particularly vulnerable. Moreover, pre-maturity is a major risk for bacterial meningitis. In addition, GBS can evade the immune system through survival inside macrophages. GBS produces an orange carotenoid pigment with free‐radical scavenging properties that neutralise hydrogen peroxide, superoxide, hypochlo-rite, and singlet oxygen and thereby provide a shield against several elements of phagocyte ROS killing (Liu et al., 2004). Gene expression of the NADH peroxidase is also highly upregulated in GBS‐infected macrophages, most likely to counteract the production of ROS. This results in increase intracellular survival of GBS (Korir et al., 2018). To perform respiration, GBS contains two enzymes in its respiratory chain, a type 2 NADH dehydrogenase (NDH‐2) and a cytochrome bd oxygen reductase. The absence of NDH_{‐2 results in the loss of GBS} virulence in a mouse model of GBS invasive disease (Lencina et al., 2018). NADH_{‐dependent enzymes therefore constitute potential drug} targets for GBS meningitis.

Following its survival within the circulation, GBS can interact with brain endothelial cells, resulting in BBB invasion and meningitis. Adhe-sion to brain endothelial cells is mediated by bacterial lipotechoic acid (LTA), while other bacterial adhesins interact with basement mem-brane components as the pilus protein PilA, the serine_{‐rich repeat} pro-teins (Srr), the streptococcal fibronectin‐binding protein (Sfb), and the alpha C protein (ACP), which binds respectively with collagen, plasmin-ogen, fibronectin, and to glycosaminoglycan (Tazi et al., 2012). ST‐17 strains specifically express Srr2, which binds plasminogen and plasmin, and a surface protein called hypervirulent GBS adhesin (HvgA), which binds to glycosaminoglycans. These adhesins increase bacterial sur-vival to phagocytic killing, and more interestingly, promote bacterial persistence in a murine model of meningitis, suggesting that hijacking ligands of the host coagulation system by Srr2 contribute to GBS

dissemination and invasiveness and ultimately to meningitis (Six et al., 2015). More recently, a GBS cell wall_{‐anchored adhesin that binds} host plasminogen, called PbsP for Plasminogen binding surface Pro-tein, was shown to be required for brain invasion by hypervirulent CC17 Group B streptococci (Buscetta et al., 2016; Lentini et al., 2018). In addition, a GBS cell wall protein called BspC protein (group B Streptococcus secreted protein) seems also required to promote GBS adherence to brain endothelial cells by interacting with vimentin, an intermediate filament protein highly expressed in endothelial cells (Deng et al., 2019). Animal models and in vitro culture systems have shown that GBS can invade brain endothelial cells in favour of the transcellular route to cross the BBB without any evidence of disrup-tion of the intracellular tight juncdisrup-tions (Kim, 2008). However, GBS infection of brain endothelial cells also results in the induction of the host transcriptional repressor Snail1, which impedes expression of the tight junction genes ZO_{‐1, claudin 5, and occludin. Snail1} expres-sion was sufficient to facilitate tight junction disruption, promoting BBB permeability to allow bacterial passage through a paracellular pathway (Kim et al., 2015). In addition, GBS expresses a pore forming β‐haemolysin/cytolysin (β‐h/c), which is cytolytic for human brain endothelial cells and may contribute to BBB disruption (Cutting et al., 2014). Disruption of barrier integrity may then result from bac-terial passage through a paracellular route, cellular damages induced by secreted cytotoxins, and/or induction of host inflammatory pathways.

Currently, no GBS vaccine is available. In 2014, the World Health Organization summoned the first meeting regarding the development of GBS vaccines (Giersing, Modjarrad, Kaslow, & Moorthy, 2016). Recently a trivalent polysaccharide conjugate vaccine (PCV) has been developed, and a phase Ib/2 clinical trial (NCT01193920) in neonates born to vaccinated women has been performed (Madhi et al., 2017). The level of maternal specific antibodies against capsular polysaccha-ride of GBS in these infants was higher at birth, showing that maternal immunization could be protective. In 2017, a phase 1/2 clinical trial evaluating a new pentavalent vaccine (NCT03170609) has started.

1.2.1

_{Escherichia coli}

E. coli is an extracellular facultative anaerobic and coliform Gram neg-ative bacillus. It is the second cause of meningitis in newborns (Ouchenir et al., 2017). Rectal carriage of E. coli is up to 50%, and about 70% of neonates born to E.coli_{‐colonised mothers become} colonised by their mother's without necessarily causing pathogenicity. In about 80% of cases, E. coli strains responsible for meningitis possess the K1 capsular polysaccharide antigen, which gives protection from immune attack.

The development of E. coli K1 meningitis is preceded by gastroin-testinal mucosa invasion and high_{‐level bacteraemia (Xie, Kim, &} Kim, 2004). Bacteraemia did not increase in PMN‐depleted mice sug-gesting that PMNs may provide a niche allowing bacterial multiplica-tion (Mittal & Prasadarao, 2011). In addimultiplica-tion, selective deplemultiplica-tion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis, suggesting that these cells could also provide

a niche for bacterial proliferation (Sukumaran, Shimada, & Prasadarao, 2003). The expression of the outer membrane protein A (OmpA) is critical for entry and survival inside both PMNs and macrophages. OmpA interacts with gp96, an endoplasmic paralog of heat shock pro-tein 90, which may gain extracellular access after PMN activation or infection. Interaction of OmpA with surface expressed gp16 sup-presses the release of ROS, indicating that E. coli prevents the antimi-crobial activity of PMNs. In macrophages, OmpA binds to N_‐glycan moieties on the IgG binding receptor CD64. CD64 knockout mice are not susceptible to E. coli meningitis, while adoptive transfer of wild‐type macrophages into these mice is sufficient to restore their vulnerability to this bacterial infection (Krishnan et al., 2014). Several in vivo observations suggest that high level of bacteraemia facilitates interaction with cerebral capillaries and is a prerequisite for E. coli entry into the brain (Kim, 2012). Several microbial factors contributing to invasion of human brain endothelial cells by E. coli have been described, which include OmpA, FimH, NlpI, IbeA, IbeB, IbeC, and CNF1 (Kim, 2012). These microbial factors use specific host cell recep-tors and signalling molecules to induce actin cytoskeleton rearrange-ment in brain microvascular endothelial cells and the formation of microvillous protrusions that facilitate E. coli K1 internalisation (Kim, 2008). Bacteria stored in endothelial vacuoles may then reach the sub-arachnoid space after release at the endothelial basal membrane. OmpA binds to brain endothelial cells for invasion via a lectin_‐like activity specific to a carbohydrate epitope (chitobiose) attached to asparagine_{‐linked glycoproteins (Prasadarao, Wass, & Kim, 1996).} Chitooligomers treatment prior to E. coli infection of newborn rodents could prevent the occurrence of meningitis. Subsequent studies have shown that OmpA interacts with two N‐glycosylation sites on Ecgp96 (the_{β‐form of gp96) present in human brain endothelial cells to} pro-mote binding to and invasion of the cells (Prasadarao et al., 2003), whereas Contactin_{‐associated protein 1 (Caspr1), a neuronal} trans-membrane protein also expressed at the cell surface of brain endothe-lial cells, is a host receptor for IbeA (Zhao et al., 2018). Genetic ablation of endothelial Caspr1 and blocking IbeA–Caspr1 interaction prevent translocation of the BBB by E. coli and reduce pathogenesis. This receptor might be a useful target for prevention or therapy of E. coli meningitis.

In addition, interaction of the bacterial adhesin FimH with CD48 and α7 Nicotinic Acetylcholine Receptor induces intracellular calcium signal-ling and cell cytoskeleton remodelsignal-ling promoting PMN transmigration across a brain endothelial cell monolayer in vitro, suggesting a potential synergy in FimH‐induced E. coli K1 invasion and PMN migration across the BBB (Liu et al., 2019). Finally, it has been shown in vitro that bacterial haemolysin‐co‐regulated protein 1 (Hcp1), a component of the Type VI secretion system of E. coli K1, can be injected in the cytoplasm of human brain endothelial cells and induces apoptosis (Zhou et al., 2012). A recent work aiming at identifying brain endothelial cell targets for Hcp1 found “IQ motif containing GTPase activating protein 1” (IQGAP1), a scaffold protein involved in regulating activation of various signalling processes, including MAPK signalling pathway (Zhao et al., 2017). The use of the MAPK inhibitor U0126 demonstrated protective effects against E. coli meningitis in a mouse model with significant decrease in neutrophil

recruitment and in the production of inflammatory cytokines, such as IL‐ 1_{α, IL‐1β, IL‐6, and TNF‐α among U0126 treated mice (Zhao et al., 2017).} Among host cell signalling molecules hijacked by E. coli to promote brain penetration, there is epidermal growth factor receptor (EGFR) tyrosine kinase receptor. OmpA, FimH, or NlpI‐mediated adhesion all induce acti-vation of sphingosine 1_{‐phosphate (S1P) allowing EGFR activation and} penetration of the BBB in vitro and in vivo (Wang et al., 2016). It is inter-esting to note that other bacterial pathogens, such as Neisseria meningitidis and Haemophilus influenza, also target EGFR family members through different mechanisms to facilitate their infection of host cells (Hoffmann, Eugene, Nassif, Couraud, & Bourdoulous, 2001; Mikami et al., 2005).

It remains unclear whether brain damages associated with E. coli are due to direct interaction with neuronal or glial cells or due to a col-lateral effect of an inflammatory response, one does not exclude the other. Currently, there is no available licensed vaccine for E. coli.

2

_{C O N C L U S I O N}

Significant progresses have been made in recent years to identify pathophysiological mechanisms contributing to host–bacterial interac-tions during bacterial meningitis. Those include the characterisation of pathways used by these pathogens to cross mucosa, survive in the blood and promote innate immune response, and/or immune escape, along with the identification of ligand or receptor interactions used by these bacterial species to cross the brain barriers. These studies already helped the development of effective therapies. In the light of the low efficacy of current vaccines and antibiotic resistance, targeting bacterial adhesins and/or their host receptor and associated signalling events represent particularly appealing therapeutic strategies to reduce the heavy toll of bacterial meningitis.

A C K N O W L E D G E M E N T S

L.LG. was supported by a doctoral fellowship from the Fondation pour la Recherche Médicale. This work was supported by collaborative research grants from the Agence Nationale de la Recherche to XN and SB (ANR_{‐14‐CE14‐0010‐01 and ANR‐14‐IFEC14‐0006) and by} an ANR grant (ANR‐15‐CE15‐0002) to MC. SB, MC and XN are sup-ported by INSERM, CNRS, Université Paris Descartes and the Fondation pour la Recherche Médicale.

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

The authors have no conflict of interest to declare

O R C I D

Mathieu Coureuil https://orcid.org/0000-0001-7655-9685

Sandrine Bourdoulous https://orcid.org/0000-0003-2852-4765

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How to cite this article: Le Guennec L, Coureuil M, Nassif X, Bourdoulous S. Strategies used by bacterial pathogens to cross the blood–brain barrier. Cellular Microbiology. 2020;22:e13132.