TY - JOUR
AU - Schubert-Unkmeir, Alexandra
AB - Abstract Neisseria meningitidis is one of the most common aetiological agents of bacterial meningitis, affecting predominantly children and young adults. The interaction of N. meningitidis with human endothelial cells lining blood vessels of the blood–cerebrospinal fluid barrier (B-CSFB) is critical for meningitis development. In recent decades, there has been a significant increase in understanding of the molecular mechanisms involved in the interaction of N. meningitidis with brain vascular cells. In this review, we will describe how N. meningitidis adheres to the brain vasculature, may enter inside these cells, hijack receptor signalling pathways and alter host-cell responses in order to traverse the B-CSFB. Neisseria meningitidis; blood–cerebrospinal fluid barrier, surface adhesins and invasins, signal-transduction mechanisms, ceramide INTRODUCTION Neisseria meningitidis is an aerobic, non-spore-forming Gram-negative coccobacillus that is frequently found as an asymptomatic coloniser of the human upper respiratory tract. Under certain circumstances, N. meningitidis may penetrate the mucosal membrane, enter the bloodstream and cause severe septicaemia and/or meningitis. Colonisation is necessary for survival and propagation of this species, and also represents the first step in the disease process. For colonisation of the human nasopharynx, the microorganism must adhere to the mucosal surface, utilise locally available nutrients and evade the human immune system. Neisseria meningitidis expresses a wide range of structures and molecules that facilitate adhesion. These structures also allow the bacterium to hijack host-cell receptors and signalling pathways resulting in penetration and/or disruption of cellular barriers, leading to disease progression. Due to the human-restricted tropism of N. meningitidis, it has been difficult to establish suitable animal models for studying the mechanisms by which meningococci colonise and transverse host barriers. Yet, particular models have been developed to reflect the different stages: isolated, immortalised epithelial cells have been used to understand the process of colonisation, human whole blood and rodent models have been adapted to study survival and dissemination within the bloodstream and primary cultures of human brain microvascular endothelial cells or immortalised human microvascular endothelial cell lines have been implemented to study the subsequence events of meningococcal attachment to brain microvessels and entry into the cerebrospinal fluid barrier (CSF). In recent years, important advances have been made in understanding the molecular and cellular events especially involved in the steps of bacterial interaction with the brain endothelium. In this review, I will summarise the molecular mechanisms of vasculature interaction by N. meningitidis. Bacterial factors and receptors involved in Neisseria meningitidis interaction with cells of the B-CSFB Bacterial binding to and invasion of brain endothelial cells is a prerequisite for successful penetration into the CSF. Like most commensal and pathogenic bacteria N. meningitidis possesses a variety of adhesins and invasions that facilitate the interaction with host-cell receptors or with soluble macromolecules. Exploitation of in vitro cell culture systems has proven to be a valuable tool to study bacterial adhesion/invasin receptor interaction in detail and to decipher involved signalling cascades. Several in vitro cell culture models have been implemented for N. meningitidis research and will be introduced in the following section. In vitro cell culture models to study the interaction of Neisseria meningitidis with cells of the B-CSFB As N. meningitidis is an exclusively human pathogen, it has proved difficult to establish suitable animal models for studying the mechanisms by which meningococci colonise and transverse host barriers, including the blood–CSF barrier (B-CSFB). Most of the knowledge of the infection pathway of N. meningitidis has been derived from studies with organ cultures, primary cell cultures or immortalised cell lines. A number of cell lines have been used to study the interaction of N. meningitidis with brain capillaries, including human bone-marrow endothelial cells (Schweitzer et al.1997; Hoffmann et al.2001; Lambotin et al.2005), the brain microvascular endothelial cell line hCMEC/D3 (Weksler et al.2005; Coureuil et al.2009, 2010; Lecuyer, Nassif and Coureuil 2012; Weksler, Romero and Couraud 2013; Bernard et al.2014), human brain microvascular endothelial cells (HBMEC) (Stins, Gilles and Kim 1997; Stins, Badger and Kim 2001; Unkmeir et al.2002; Sokolova et al.2004; Simonis et al.2014) and BB19 cells (Prudhomme et al.1996; Bernard et al.2014). The ideal cell line should retain the endothelial nature, such as tubule formation, surface expression of intercellular-adhesion molecule 1 (ICAM-1), vascular-cell adhesion protein 1 (VCAM), E-selectin and CD36 and should be positive for factor VIII-related antigen, or von Willebrand factor. HBMEC were obtained by immortalisation of human cerebral endothelial cells using SV40 large T antigen (Stins, Gilles and Kim 1997; Stins, Badger and Kim 2001) and has been widely used to study the interaction of a number of meningitis-causing pathogens, including bacteria, fungi and even parasites (for a review, see Kim 2008). The human bone-marrow endothelial cell line was isolated from bone-marrow capillaries that maintained most of the characteristics of primary endothelial cells during in vitro culture (Schweitzer et al.1997). hCMEC/D3 is a fully differentiated human brain endothelial cell line derived from human temporal lobe microvessels and recapitulates the major phenotypic features of the B-CSFB (Weksler et al.2005; Weksler, Romero and Couraud 2013). BB19 is a human brain endothelial cell line transformed by papilloma virus and was first applied for the analysis of the cytoadherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells (Prudhomme et al.1996). Exploitation of in vitro cell culture systems has proven to be a valuable tool to study host-cell interaction for over a century, but as any tool, is subject to limitations, artefacts and misleading results when removed from physiological context without validation or justification. While none of these models is ideal, they have revealed that N. meningitidis is capable to hijack host-cell receptors and signalling pathways to overcome the B-CSFB. Therefore, our understanding of the molecular mechanisms involved in the interaction of N. meningitidis with brain vascular cells has greatly increased due to the use of these various in vitro cell-culture models. Bacterial ligand–receptor interactions Neisseria meningitidis possesses a variety of determinants that contribute to adhesion and penetration of the B-CSFB, including pili, outer membrane proteins, lipopolysaccharides and a number of so-called minor adhesion proteins. The following section reviews the adhesion process and highlights the bacterial ligand–host-cell receptor interaction that may determine the tropisms to brain endothelial cells. Type IV pili Meningococcal type IV pili are polymeric filaments found in a variety of Gram-negative bacteria (Strom and Lory 1993). In addition to mediating contact between N. meningitidis and eukaryotic cell surfaces, type IV pili are involved in bacterial movement, also known as twitching motility in the case of Neisseria, and transformation competence. The pili mediate bacterial aggregation and trigger the initiation of signalling events in host cells. Meningococci are capable to produce two structurally distinct types of pili (class I and class II), whereas the related species N. gonorrhoeae, the causative agent of gonorrhoea, only produce class I pili. The major component of type IV pili is the major pilin, PilE, which assembles into a helical fibre, with the alpha helix hidden inside the centre of the structure. Recently, the crystal structure of the N. meningitidis major pilin PilE has been described (Kolappan et al.2016), and crystal structure and cryoEM reconstruction revealed the molecular arrangement of the N-terminal α-helices in the filament core (Kolappan et al.2016). Genetic screens have revealed that building a fully functional type IV pilus involves more than 20 proteins of which 15 are essential for type IV pilus biogenesis (Carbonnelle et al.2006; Brown et al.2010). The pilin is synthesised as a preprotein and the inserted into the cytoplasmatic membrane by the Sec machinery. The leader sequence of the prepilin is then proteolytically cleaved and the mature pilin N-methylated by the inner membrane prepilin peptidase PilD (Strom, Nunn and Lory 1993). Mature pilins are then extracted from the cytoplasmatic membrane, assembled in fibres in the periplasm by the type IV pilus assembly machinery, which comprises at least the ATPase PilF (Freitag, Seifert and Koomey 1995) and the integral membrane protein PilG (Tonjum et al.1995). Finally, pilus fibres exit the outer membrane through a pore composed of the PilQ secretin (Wolfgang et al.2000; Collins et al.2001). This system is driven by ATPases, which provide the energy for the extension or retraction of the type IV pili. In addition to PilE, type IV pili in N. meningitidis also contain so-called minor pilins, PilX, PilV and ComP (Helaine et al.2005; Brown et al.2010), that share structural features with the major pilin and are supposedly similarly assembled within the filaments. However, a recent study suggests the location of the minor pilins PilV and PilX in the bacterial periplasm rather than along pilus fibres (Imhaus and Dumenil 2014) and their participation in the initiation phase of pilus biogenesis. Each minor pilin modulates type IV pili-linked properties, including competence for DNA transformation (ComP), bacterial aggregation (PilX) and indirect adhesion (PilX) and signalling (PilV) to human cells (Wolfgang et al.1998; Helaine et al.2005; Brissac et al.2012). Although pilus expression is crucial for the initial adhesion of pathogenic Neisseria strains to host cells, the receptor active in this process has been unclear and subject for research and controversy discussion for many years. CD46 (membrane cofactor protein) was described as a proposed host-cell receptor for type IV pili of N. gonorrhoeae (Källström et al.1997); however, the role of CD46 as a host-cell receptor is controversial and yet no direct interaction between CD46 and any pilus component, such as the PilC adhesin, has been demonstrated. Specific downregulation of CD46 expression in human epithelial cell lines by RNA interference did not alter the binding efficiency of piliated N. gonorrhoeae strains, although other CD46-dependent processes, such as measles virus infection and C3b cleavage, were significantly reduced. (Kirchner, Heuer and Meyer 2005). Moreover, it was repeatedly reported that pretreatment with anti-CD46 antibodies could not block pilus-mediated adherence of N. gonorrhoeae to epithelial cells (Edwards et al.2002; Gill et al.2003; Edwards and Apicella, 2005; Kirchner, Heuer and Meyer 2005). In addition, no correlation between levels of CD46 expression on the cell surface of different cell lines and the degree of adhesion of piliated Neisseriae could be observed (Tobiason and Seifert 2001; Kirchner, Heuer and Meyer 2005), indicating that CD46 does not act as a classic receptor for type IV pili. Recently, CD147 (basigin, extracellular matrix metalloproteinase inducer (EMMPRIN)) was described a receptor for meningococcal type IV pili targeted on brain endothelial cells (Bernard et al.2014). CD147 is a cell surface glycoprotein that belongs to the immunoglobulin superfamily. Bernard et al. (2014) showed that N. meningitidis utilises CD147 for type IV pili-dependent adhesion to endothelial cells and demonstrated the central role of this glycoprotein for vascular colonization of pathogenic meningococci. They demonstrated that type IV pilus-mediated adhesion relied on the major pilin PilE and the minor pilin PilV. Interfering with this interaction blocked binding of meningococci to human endothelial cells in vitro and, importantly, also prevented colonisation of vessels in human brain-tissue explants ex vivo. Additionally, PilE- and PilV-dependent colonisation of N. meningitidis to endothelial vessels was verified in vivo using a model of severe combined-immunodeficiency mice grafted with human skin (Bernard et al.2014). However, the identification of CD147 was based mainly on N. meningitidis strain 2C4.3 (formerly 8013/clone12) and its pilin. This isolate was selected for its high adhesiveness after repeated passages on epithelial cells (Nassif et al.1993). To verify the role of CD147 for type IV pili-dependent adhesion to endothelial cells, more data may be required including clinical isolates and not only laboratory-established strains. Opacity proteins: Opa and Opc Type IV pili traverse the polysaccharide capsule, the outermost layer of N. meningitidis, and are generally regarded as the most important adhesion of fully encapsulated meningococci. Though outer membrane proteins are partially masked by the capsule, they also efficiently support adhesion and invasion of eukaryotic cells, especially on cells exhibiting high degrees of receptor density, as would be induced under inflammatory conditions and/or lateral-receptor aggregation (Virji et al.1996a,b; Hardy et al.2000; Unkmeir et al.2002; Bradley et al.2005). The outer membrane proteins comprise the colony opacity-associated proteins Opa and Opc. Opa proteins are structurally highly variable proteins and there are ∼4 to ∼5 loci encoding Opa proteins in N. meningitidis (opaA, opaB, opaD and opaJ). Opa proteins consist of eight transmembrane β-strands and four surface-exposed loops. Several studies have demonstrated their important role in tight adhesion and entry into host cells (Virji et al.1993, 1996a,b; Wang et al.1998). This process is mediated mainly by interaction with carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) found in a wide variety of human tissues (Virji et al.1996a,b; Chen et al.1997). CEACAM receptors are differentially expressed by epithelial and endothelial. Moreover, the level of glycosylation of CEACAM receptors may vary, depending on their cell type and differentiation state, and multiple glycoforms of the same protein have been isolated. The particular role of CEACAM1, the most commonly expressed member of the CEACAM family, in Neisseria pathogenicity has been documented for epithelial cells. Opa binding to CEACAM1 involves two of the four extracellular loops of the Opa protein and the unglycosylated face of the NH2-terminal Ig variable-like domain of CEACAM1 (Virji et al.1996a,b; Bos, Hogan and Belland 1999; Bos et al.2002). However, since CEACAM receptors are differentially expressed by epithelial and endothelial, the Opa/CEACAM interaction cannot simply be transferred to endothelial cells. There is indeed limited information regarding the contribution of Opa/CEACAM interaction during meningococcal infection of endothelial-vessel cells. Endothelial cells such as human umbilical vein endothelial cells (HUVECs) express little CEACAM receptor in vitro; however, a substantial upregulation of CEACAM1 expression can be achieved following treatment with the proinflammatory cytokine tumour necrosis factor (TNF)-α (Muenzner et al.2001). Interestingly, binding of meningococcal Opa proteins to CEACAM1 was described in a study using HBMEC; however, this study was performed using HBMEC that were transfected to express human CEACAM proteins (Voges et al.2012). To our knowledge, a determination of meningococcal Opa-CEACAM1 interaction—for example, after TNF-α treatment of human brain vessels—has not been undertaken. The outer membrane protein Opc is particularly implicated in host-cell invasion of endothelial cells (Virji et al.1992, 1993, 1995; Virji, Makepeace and Moxon 1994; Unkmeir et al.2002). Opc is a β-barrel protein, with five surface loops encoded by a single gene (opcA) and, unlike the Opa proteins, is antigenically stable. Its expression is controlled at the transcriptional level by the length of a polycytidine stretch within the opcA-promoter region (Sarkari et al.1994) (the number of nucleotide repeats determines promotor strength and binding efficacy of the RNA polymerase). Opc is expressed by several virulent N. meningitidis lineages, but is absent from certain epidemic clones [ET-37/ST-11 clonal complex (cc)] and a few random endemic isolates (Sarkari et al.1994). Interestingly, two epidemiological studies reported outbreaks where meningococcal strains of the ST-11 cc caused severe sepsis with fatal outcomes, but rarely meningitis (Kriz, Vlckova and Bobak 1995; Whalen et al.1995), suggesting Opc as a major candidate that enhances the ability of bacteria to bind to brain endothelial cells and cause meningitis (Unkmeir et al.2002). Both Opa and the Opc protein can bind directly to components of the extracellular matrix (ECM) and serum proteins, such as vitronectin or fibronectin. Additionally, they may indirectly bind to fibronectin and vitronectin via heparin, because both fibronectin and vitronectin are heparin-binding proteins. By binding to fibronectin or vitronectin, bacterial adhesins can also target proteoglycans. The tight association of Opc to vitronectin and/or fibronectin mediates binding of the bacteria to their cognate receptor, endothelial αVβ3 integrin (vitronectin receptor) (Virji, Makepeace and Moxon 1994; Sa, Griffiths and Virji 2010) and/or α5β1-integrin (fibronectin receptor) (Unkmeir et al.2002) on brain-vessel cells. Whereas the vitronectin receptor was shown to be the main receptor targeted by N. meningitidis on HUVECs, the fibronectin receptor is targeted on HUVECs and on human brain vessels as demonstrated on HBMEC (Unkmeir et al.2002). Detailed analysis revealed that fibronectin is bound to a lesser extent to Opc than vitronectin (Sa, Griffiths and Virji 2010). Opc binds preferentially to the activated form of human vitronectin, and binding involves the connecting region of vitronectin and requires sulphated tyrosines (Sa, Griffiths and Virji 2010). Interestingly, various pathogens utilise ECM proteins to enhance adhesion and invasion to host cells or even as a target for degradation in order to breach cellular barriers (reviewed in Schwarz-Linek, Hook and Potts 2004; Singh et al.2012). Other adhesins Other adhesins such as the neisserial adhesion and penetration protein (App) (Serruto et al.2003), neisserial adhesin A (nadA) (Capecchi et al.2005), Neisseria hia/hsf homologue (NhhA) (Scarselli et al.2006), the adhesin complex protein (ACP) (Hung, Heckels and Christodoulides 2013), the autotransporter meningococcal serine protease A (MspA), fructose-1, 6-bisphosphate aldolase (FBA) (Tunio et al.2010a,b), glyceraldehyde 3-phosphate dehydrogenase (GapA-1) (Tunio et al.2010a,b) or T-cell stimulating protein A (TspA) (Oldfield et al.2007) all mediate adhesion to eukaryotic cells and some of them have been shown to be involved in endothelial cell interactions: the 13-kDa meningococcal ACP, for example, mediates association to endothelial cells, independent of capsulation (shown on HUVECs) (Hung, Heckels and Christodoulides 2013). The autotransporter MspA was cloned in Escherichia coli and was demonstrated to support adhesion HBMEC (Turner et al.2006), and the classical housekeeping genes GapA-1 and FBA were found to be localised to the surface, contributing to bacterial adhesion to HBMEC also in a capsule-independent process, and suggesting a role in the pathogenesis of meningococcal infection (Tunio et al.2010a,b). However, the recognition receptors of these adhesins remain to be determined. Link between metabolism and host-cell interaction Recently, new high-throughput technologies were employed to decipher our knowledge of gene function and to reveal the relationship between the genotype and phenotype. For example, massive parallel sequencing has been combined with traditional transposon mutagenesis techniques referred to as transposon sequencing (Tn-seq), high-throughput insertion tracking by deep sequencing (HITS), insertion sequencing (INSeq) and transposon-directed insertion site sequencing (for an overview, see van Opijnen and Camilli 2013). HITS was applied to a library of ∼70 000 N. meningitidis mutants (Capel et al.2016). In in vitro models, including endothelial and epithelial cells infected under flow conditions to reflect physiological micromechanical environment, a total of 383 genes and eight intergenic regions containing small non-coding RNA were identified that either participated in bacterial growth or were required for host-cell colonisation. In this study, 108 genes were found to be necessary for colonisation of both human cell types, endothelial and epithelia cells, and 29 genes were only selected in the endothelial cell model (Capel et al.2016). Interestingly, among the genes necessary for endothelial cell colonisation not only genes encoding for surface-exposed adhesins were upregulated but also a majority of genes that are involved in metabolic pathways (e.g. glycolysis, tricarboxylic acid cycle, PP, KDPG pathways). During the last years, it has become evident that bacteria need a further additional requirement for fitness and success in addition to ‘classical’ virulence factors. A simple additional premise is that bacteria are able to obtain nutrients and ions at the infection site. As a consequence, bacterial pathogens have evolved efficient nutrient retrieval strategies to optimise their metabolism and maximise the acquisition of essential ions and energy sources during infection. For example, to infect brain endothelial vessels, N. meningitidis has to survive and disseminate within the bloodstream. Therefore, the bacterium needs to adapt to survive and grow in this environment. Interestingly, an ex vivo model of human whole blood infection has been used to mimic the septic phase of meningococcal disease and revealed new insights how N. meningitidis adapts to permit survival and growth during bacteraemia (Echenique-Rivera et al.2011). By analysing the transcriptional response in human whole blood, this study showed that the bacterium for example adapts its energy metabolism during incubation in blood, with genes in both aerobics and anaerobic metabolic pathways being regulated. The ferric-uptake regulator protein (Fur) represented a major regulator of adaptation in human blood, and genes involved in iron acquisition and storage were one of the main functional classes activated. Interestingly, genes encoding for adhesins were also found to be upregulated, such as the Opa proteins, Opc or MspA, suggesting that in addition to their main role in interaction with host cells, these factors might also be involved in the interaction with blood cells or in the survival in whole blood (Echenique-Rivera et al.2011). Taken together, data from these studies further support the concept of nutritional virulence and metabolic adaptation of meningococci upon adhesion to human cells (Schoen et al.2014). Subversion of host-cell proteins at the site of meningococcal adhesion Following initial attachment, successful colonisation of endothelial cells requires bacterial aggregation and microcolony formation. When adhering to endothelial cells, N. meningitidis induces the local formation of protrusions resembling epithelial microvilli structures (Eugene et al.2002), which surround the bacteria and initiate their internalisation within vacuoles (Eugene et al.2002). These structures increase on the cell-membrane surface to facilitate bacterial adhesion and contribute to resistance of the growing microcolonies against shear stress in the bloodstream (Mairey et al.2006). Importantly, formation of the protrusions was also observed ex vivo in sections of a choroid plexus capillary from a postmortem examination of a paediatric patient who died of fulminant meningococcaemia (Nassif et al.2002). The formation of membrane protrusions results from the organisation of a molecular complex, also called a ‘cortical plaque’, underneath bacterial microcolonies (Merz, Enns and So 1999). Cortical plaques are enriched from transmembrane proteins such as CD44, ICAM1, VCAM1, the epidermal growth factor receptor as well as from the molecular-linker proteins ezrin and moesin, and from the localised polymerisation of cortical actin (Merz and So, 1997; Merz, Enns and So 1999; Eugene et al.2002). Cortical plaque formation was initially described after pilus-mediated adhesion to infected epithelial cells, but can also be induced on human brain microvascular cells. However, whereas pilus-mediated adhesion induces apparently similar cortical plaques in epithelial and endothelial cell lines, the signalling pathways leading to cortical plaques as well as the consequences of this formation have been demonstrated to be strikingly different for the two cell types (Lecuyer, Nassif and Coureuil 2012). Differences include the activation of the β2-adrenergic receptor (AR)/β-arrestin pathway as well as recruitment of polarity complex and cell junction proteins, which are only found after contact of N. meningitidis with endothelial cells and not during infection of epithelial cells, and will be outlined in the following section. In brain endothelial cells, the formation of cortical plaque results from the recruitment and activation of the β2-AR by type IV pili (Coureuil et al.2010). β2-AR belongs to the large and diverse family of G-protein-coupled receptors. The interaction of meningococci with the N-terminal domain of β2-AR most likely modifies receptor conformation, resulting in activation of β-arrestin-mediated signalling pathways (Coureuil et al.2010). β-Arrestin translocation in turn leads to activation of c-Src, a non-receptor tyrosine kinase that phosphorylates diverse substrates and finally mediates formation of actin-rich cellular protrusions. Additionally, β-arrestin translocation leads to the accumulation of β-arrestin-interacting proteins, such as VE cadherin and p120 catenin, into cortical plaques underneath bacterial microcolonies. Besides recruitment of molecular-linker proteins, mem-brane-integral proteins and β-arrestin-interacting proteins, N. meningitidis also modulates the localisation of polarity complexes proteins and tight junction expression in brain endothelial cells (Coureuil et al.2009; Schubert-Unkmeir et al.2010). Endothelial cells in brain capillaries are highly specialised and characterised by the presence of junctional complexes between cerebral endothelial cells. The junctional complexes comprise tight junction and adherens junctions, consisting of transmembrane proteins and cytoplasmic plaque proteins. Whereas the former proteins physically associate with their counterparts on the plasma membrane of adjacent cells, the latter provide a link between transmembrane tight- and adherens-junction proteins and the actin cytoskeleton and participate in intracellular signalling (reviewed in Stamatovic, Keep and Andjelkovic 2008). Transmembrane proteins of the tight junction include occludin, claudins and junctional adhesion molecule (JAM)-A, JAM-B and JAM-C (Citi and Cordenonsi, 1998; Martin-Padura et al.1998; Gonzalez-Mariscal et al.2003). Occludin was one of the first tight junction transmembrane proteins described. Structurally, occludin contains two equal extracellular loops, four transmembrane domains and three cytoplasmic domains: one intracellular short turn, a small N-terminal domain and a long carboxyl (C-) terminal (Furuse et al.1993; Li et al.2005; Nusrat et al.2005). Interestingly, the tight junction protein occludin is targeted during meningococcal infection: in vitro assays showed that meningococcal infection of brain endothelial cells resulted in proteolytic cleavage of occludin (Schubert-Unkmeir et al.2010). Cleavage is mediated by matrix metalloproteinase (MMP)-8 and occludin is cleaved to a lower molecular weight 50-kDa protein in infected cells, suggesting that cleavage occurs within the first extracellular loop. As a consequence of proteolytic cleavage, occludin disappears from the cell periphery, resulting in weakening of the barrier property of the microvascular brain endothelial cells (Schubert-Unkmeir et al.2010) (Fig. 1). Figure 1. View largeDownload slide Schematic representation of the steps of N. meningitidis interaction with brain endothelial cells and activated signalling pathways. After initial binding to host cells, meningococci proliferate, form small aggregates (microcolonies) at the site of attachment on the cell surface and induce organisation of cortical plaque structures along with accumulation of the transmembrane receptor CD147 (1). In parallel, N. meningitidis stimulates the β2-adrenoceptor(AR)/β-arrestin (2) signalling pathway in brain endothelial cells, which activates c-Src and results in ectoptic activation of the polarity complex Par3/Par6/PKCζ (3). Recruitment of tyrosine kinases (c-Src and FAK) results in phosphorylation and activation of the cortactin/Arp2/3 complex and cytoskeletal rearrangement. In addition, the tight junction protein occludin is targeted during meningococcal infection and cleaved, which involves MMP-8 activity (adapted from and according to Coureuil et al.2009; Lemichez et al.2010; Slanina et al.2010; Slanina et al.2012; Bernard et al.2014). TJ, tight junction; AJ, adherens junction; Des, desmosomes. Figure 1. View largeDownload slide Schematic representation of the steps of N. meningitidis interaction with brain endothelial cells and activated signalling pathways. After initial binding to host cells, meningococci proliferate, form small aggregates (microcolonies) at the site of attachment on the cell surface and induce organisation of cortical plaque structures along with accumulation of the transmembrane receptor CD147 (1). In parallel, N. meningitidis stimulates the β2-adrenoceptor(AR)/β-arrestin (2) signalling pathway in brain endothelial cells, which activates c-Src and results in ectoptic activation of the polarity complex Par3/Par6/PKCζ (3). Recruitment of tyrosine kinases (c-Src and FAK) results in phosphorylation and activation of the cortactin/Arp2/3 complex and cytoskeletal rearrangement. In addition, the tight junction protein occludin is targeted during meningococcal infection and cleaved, which involves MMP-8 activity (adapted from and according to Coureuil et al.2009; Lemichez et al.2010; Slanina et al.2010; Slanina et al.2012; Bernard et al.2014). TJ, tight junction; AJ, adherens junction; Des, desmosomes. In addition to targeting tight junction proteins, N. meningitidis triggers recruitment of the polarity complex Par6/Par3/PKCζ (Coureuil et al.2009), which is followed by the delocalisation of junctional proteins from the intercellular junctions (Coureuil et al.2009). The PAR complex is composed of two scaffold proteins, PAR6 and PAR3, and an atypical protein kinase C and is crucial to the formation of apical junctions such as tight junctions in mammalian cells. After meningococcal induced formation of ‘cortical plaques’, the ectoptic activation of the polarity complex Par3/Par6/PKCζ underneath the meningococcal microcolony leads to abnormal recruitment of adherens junctions proteins (e.g. VE-cadherin and p120-catenin). Of importance, proteins of the cellular junction have been shown to be recruited from the existing pool of proteins present at the cell–cell contact. Subversion of sphingolipids during meningococcal infection Bacterial binding and subsequent uptake by host cells not only involves binding to specific ligand receptors, but also requires a re-organisation of receptor and signalling molecules in the cell membrane. Recent studies indicated that specialised domains of the cell membrane called rafts are central for the spatial organisation of receptors and signalling molecules. Biological membranes of eukaryotic cells are dynamic structures and composed of large amounts of sphingolipids and cholesterol in addition to glycerophospholipids, which predominantly localise to the outer leaflet of the cell membrane. Sphingomyelin is the most prevalent membrane sphingolipid and is composed of a hydrophobic ceramide moiety and a hydrophilic phosphorylcholine head group (Futerman and Hannun 2004). Ceramide also forms the backbone of other complex sphingolipids, such as cerebrosides and gangliosides. Under certain stress conditions or the induction of inflammatory cytokines, sphingomyelin is hydrolysed by sphingomyelinases, which cleave the phosphodiester bond of sphingomyeline releasing the phosphorylcholine head group to generate ceramide. Sphingomyelinases are characterised by their pH optimum for enzymatic activity, into alkaline, neutral and acid sphingomyelinases (ASM) (Marchesini and Hannun, 2004). They are known to be localised in different compartments within the cells, with the ASM being found within lysosomes. Recent published data demonstrated that N. meningitidis is capable of activating ASM in brain endothelial cells, significantly increasing the amount of ceramide in the outer leaflet of the cell membrane (Simonis et al.2014). Mechanistically, ASM translocation and activation is triggered by binding of the bacterium to its attachment receptor, heparan sulphate proteoglycan (HSPG), followed by activation of the phosphatidylcholine-specific phospholipase C (Simonis et al.2014). Ceramide molecules tend to interact with one another to form ceramide-rich domains and, because of their biophysical properties, ceramide-rich membrane domains then fuse into extended ceramide-rich platforms (CRPs) that span from hundreds of nanometres to several micrometres. In addition to altering membrane fluidity and rigidity, CRPs sort and concentrate membrane receptors and membrane proximal signalling components, thereby amplifying cellular responses and signal transduction. Receptor recruitment within CRPs has been found for molecules such as cluster of differentiation (CD) 95 (Grassme, Schwarz and Gulbins 2001), CD40 (Grassme et al.2002), FcγII (Abdel Shakor, Kwiatkowska and Sobota 2004) or CD20 (Bezombes et al.2004) and, in case of N. meningitidis-infected brain endothelial cells, for the tyrosine kinase receptor ErbB2, an important receptor involved in bacterial uptake (Hoffmann et al.2001; Simonis et al.2014) (Fig. 2). Figure 2. View largeDownload slide Ceramide-rich platforms and N. meningitidis. Opc-expressing meningococci activate ASM in brain endothelial cells, which hydrolyses sphingomyelin (SM) to cause ceramide (cer) release and formation of extended CRPs. Herein ErbB2, an important receptor involved in bacterial uptake, is recruited. ASM activation is triggered by binding of N. meningitidis to its attachment receptor, HSPG, followed by activation of the phosphatidylcholine-specific phospholipase C (PC-PLC) (Simonis et al.2014). PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol. Figure 2. View largeDownload slide Ceramide-rich platforms and N. meningitidis. Opc-expressing meningococci activate ASM in brain endothelial cells, which hydrolyses sphingomyelin (SM) to cause ceramide (cer) release and formation of extended CRPs. Herein ErbB2, an important receptor involved in bacterial uptake, is recruited. ASM activation is triggered by binding of N. meningitidis to its attachment receptor, HSPG, followed by activation of the phosphatidylcholine-specific phospholipase C (PC-PLC) (Simonis et al.2014). PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol. Interestingly, N. meningitidis isolates belonging to the hyperinvasive sequence type (ST-11) clonal complex (cc) barely induce ASM activation and ceramide release correlating with significantly lower bacterial uptake by brain endothelial cells (Simonis et al.2014). Since ST-11 cc isolates were reported to be more likely to cause severe sepsis, but rarely meningitis, the capability of N. meningitidis to activate ASM suggests a correlation with epidemiological data and clinical presentations shown in the cell culture-based experiments. The role of sphingolipids is an entirely novel aspect in the pathogenesis of meningococcal meningitis and an excellent field for further investigation. Subversion of host-cell signalling during meningococcal infection of brain endothelial cells A beneficial strategy used by many pathogens is to interfere with the phosphorylation cascades in the intracellular signalling pathways of the host cell (Bhavsar, Guttman and Finlay 2007). Phosphorylation states are usually controlled by protein kinases and protein phosphatases. Entry into non-phagocytic cells, such as endothelial cells, involves modulation of host signal-transduction mechanisms to target the cytoskeleton, thereby facilitating bacterial uptake. Recent advances in our understanding of eukaryotic signal-transduction pathways also extended our knowledge concerning mechanisms involved in microbial invasion, intracellular trafficking and/or cytokine release from infected cells. Particularly, the identification of the signalling molecules specifically involved in rearrangement of the host-cell cytoskeleton during N. meningitidis infection has been described in several studies and is discussed in this section. The cytoskeleton of eukaryotic cells contains actin filaments, microtubules and intermediate filaments. Bacterial pathogens usually do not interact directly with actin filaments themselves, but control the polymerisation of actin filaments by modulating cellular regulators, e.g. G proteins. For example, N. meningitidis interaction with endothelial cells triggers activation of the GTPases Rho and Cdc42, which results in local polymerisation of cortical actin. Moreover, activation of the phosphoinositide 3-kinase/Rac1 GTPase signalling pathway is involved in cortactin recruitment to the site of bacterial attachment and cortactin phosphorylation (Lambotin et al.2005). Cortactin is an actin-binding protein implicated in the stabilisation and branching of actin filaments and is involved in the reorganisation of cortical actin that plays a major role in endocytosis, neuronal morphogenesis and immune-synapse formation. Interestingly, there is evidence that N. meningitidis also directly targets distinct cytoskeletal components: for example, the Opc protein has been shown to directly bind α-actinin (Sa et al.2009). This direct interaction may facilitate modulation of intracellular events to increase survival of N. meningitidis and manipulate the cytoskeleton to favour bacterial traversal across cellular barriers (Sa et al.2009); however, further direct evidence regarding the role of Opc–α-actinin interactions has yet to be elucidated. In addition to modulating G proteins, N. meningitidis is capable of interfering with phosphorylation cascades in host-cell intracellular signalling pathways. Phosphorylation states are usually controlled by protein kinases and protein phosphatases, whose functions can be altered either directly by effector proteins from bacteria or after binding to cell-membrane-anchored receptors and subsequent modulation of downstream signalling pathways. Aside from the activation of the receptor tyrosine kinases (RTK) ErbB2 and the non-RTK c-Src in a pilus-dependent manner, binding of Opc-expressing meningococci to integrins also leads to phosphorylation of two kinases in HBMEC: the focal adhesion kinase (FAK) and c-Src. As previously described, Opc-expressing N. meningitidis strains can bind to integrins via vitronectin or fibronectin, with integrin binding followed by activation of both kinases. By using pharmacological inhibitors, knockout cells and dominant-negative or constitutive-active mutants of those kinases c-Src could be identified as an important signal protein in the invasion process of N. meningitidis (Slanina et al.2010). Moreover, the FAK, which directly associates with integrins and functions in concert with Src, is also involved in meningococcal uptake (Slanina et al.2012). Similar to c-Src, pharmacological inhibition of the FAK activity by a specific inhibitor and/or overexpression of FAK mutants that were either impaired in their kinase activity or were incapable of autophosphorylation or overexpression of the dominant-negative variant resulted in reduced bacterial uptake (Slanina et al.2012). Importantly, FAK-deficient fibroblasts are invaded significantly less often by N. meningitidis. As a downstream target, cortactin is phosphorylated downstream of integrin-Src activation; therefore, a cooperative interplay between FAK, Src and cortactin enables endocytosis of N. meningitidis into host cells (Slanina et al.2012). Both c-Src and FAK are non-RTKs. In addition to activating non-RTKs, N. meningitidis can activate RTKs to modulate host-cell signalling pathways for its purposes. The use of a phosphoarray platform demonstrated activation of various RTKs and key signalling nodes, such as Akt, Erk1/2, ribosomal S6 kinase, c-Abl, and insulin receptor substrate 1 (Slanina et al.2014); however, the functional significance of RTK phosphorylation in the context of N. meningitidis interactions with brain endothelial cells remains to be elucidated. Of note, the signalling pathways activated during meningococcal entry into HBMEC may differ from those involved in the release of cytokines and chemokines: This was observed for N. meningitidis invasion of HBMEC via c-Jun kinases 1 and 2, although those kinases are not involved in cytokine release. The release of cytokines, such as interleukin (IL)-6 and IL-8, from infected HBMEC involves the p38 mitogen-activated protein kinase (MAPK) pathway (Sokolova et al.2004). Host-cell transcriptome in response to meningococcal infection Global gene expression analyses using high-density microarrays have been applied as a powerful technique of choice during the first decade of the 21st century for studying interactions between host cells and microbial pathogens or their products. A number of experimental approaches have been conducted to study host responses to N. meningitidis in different cells types, including brain endothelial cells (Wells et al.2001; Binnicker, Williams and Apicella 2003; Bonnah et al.2004; Plant et al.2004; Robinson et al.2004; Linhartova et al.2006; Schubert-Unkmeir et al.2007; Schubert-Unkmeir, Slanina and Frosch 2009). Mainly the serogroup B strain N. meningitidis MC58 was utilised by the individual research scientists to determine host-cell responses using microarrays, enabling the generated data to be more comparable despite disparities between cell lines and different array platforms. Time-course analyses were performed and provided useful information regarding temporal events related to host-cell response. Additionally, the impact of the main N. meningitidis virulence factors, including the polysaccharide capsule and the type IV pili, on the host-cell transcriptome was determined (Plant et al.2004; Linhartova et al.2006; Schubert-Unkmeir et al.2007). The analyses revealed that the capsule is a potent virulence factor capable of eliciting an early host-cell response. Furthermore, gene expression in host cells was profiled to compare the influence of whole bacteria relative to that of secreted proteins, showing that secreted meningococcal virulence factors are also important in inducing host inflammatory responses and resistance to apoptosis (Robinson et al.2004). And microarray technology was applied to study metabolic pathways in infected cells, revealing alteration of several host genes involved in iron homeostasis (Bonnah et al.2004). A comparison of gene expression in different studies revealed a stereotyped range of host immune responses in eukaryotic cells following N. meningitidis infection, with proinflammatory responses largely conserved in most cell types. In addition to inflammatory response genes, the common host response comprised genes associated with cell death/survival, adhesion molecules and the MAPK signalling pathway, as well as genes encoding proteins involved in cytoskeletal organisation. Moreover, global gene expression analysis of host cells infected with N. meningitidis extended the repertoire of differentially regulated genes to signalling pathways, which were not recognised to be involved in meningococcal host-cell interaction, such as transforming growth factor-β/Smad, Wnt/β-catenin and Notch/Jagged. A further cluster of genes differently expressed included cell adhesion genes, e.g. cadherins, integrins and ephrin (Eph) receptors, pointing to the important role of N. meningitidis on tight junction protein modulation. In particular, transcript levels of Eph receptors and Eph were altered in brain endothelial cells. The Eph family of receptors is the largest known subfamily of RTKs (Pasquale, 2008). The ligands are called ephrins. The ephrin–Eph interactions are important in development, especially in cell–cell interactions, including those involving vascular endothelial cells and specialised epithelia-regulatory cell attachment, shape and motility. However, the role of Eph receptors in meningococcal pathogenesis remains unknown. CONCLUSION The analysis of meningococcal brain endothelial host-cell interaction is most important to fully understand the pathogenesis of meningococcal meningitis and to design more effective therapies. Advances in microbial genetics, implementation of new tissue culture models and high-throughput technologies have enhanced our understanding of the molecular mechanism involved in the complex crosstalk between Neisseria meningitidis and brain endothelial cells. However, determination of the role of particular virulence factors or high-throughput screens aimed to identify new meningococcal virulence factors essential for B-CSF penetration were often performed with a single strain isolate and a particular cell line. Based on observed strain-specific variations, obtained results cannot easily be generalised and requests a more comparative analysis of multiple strains per species. Moreover, the host-cell interaction has been primarily studied using in vitro cell cultures of monolayers and cell lines derived from tumour tissue. These straightforward models are valuable to test simple parameters of host–microbe interactions and determine major microbial and host factors in adherence, invasion and transversal, but conventional in vitro cell cultures may not capture physiological and three-dimensional aspects of tissue biology that are important in assessing pathogenesis. Three-dimensional cell culture systems have gained increasing interest in drug discovery and tissue engineering due to their advantages in providing more physiologically relevant information and more predictive data and might provide a promising tool to study N. meningitidis brain endothelial cell interaction in a setting that resembles the in vivo environment. Acknowledgments The author would like to acknowledge laboratory members and researchers whose work has not been discussed in detail or reviewed elsewhere. FUNDING The work on the B-CSFB and meningococcal meningitis in the author's laboratory is supported by funding from the DFG (grant nos. SCHU2394/2-1, SCHU2394/2-2 and SCHU2394/3-1). Conflict of interest. None declared. REFERENCES Abdel Shakor AB, Kwiatkowska K, Sobota A. Cell surface ceramide generation precedes and controls FcgammaRII clustering and phosphorylation in rafts. J Biol Chem  2004; 279: 36778– 87. Google Scholar CrossRef Search ADS PubMed  Bernard SC, Simpson N, Join-Lambert O et al.  . Pathogenic Neisseria meningitidis utilizes CD147 for vascular colonization. Nat Med  2014; 20: 725– 31. Google Scholar CrossRef Search ADS PubMed  Bezombes C, Grazide S, Garret C et al.  . Rituximab antiproliferative effect in B-lymphoma cells is associated with acid-sphingomyelinase activation in raft microdomains. Blood  2004; 104: 1166– 73. Google Scholar CrossRef Search ADS PubMed  Bhavsar AP, Guttman JA, Finlay BB. Manipulation of host-cell pathways by bacterial pathogens. 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TI - Molecular mechanisms involved in the interaction of Neisseria meningitidis with cells of the human blood–cerebrospinal fluid barrier
JF - Pathogens and Disease
DO - 10.1093/femspd/ftx023
DA - 2017-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/molecular-mechanisms-involved-in-the-interaction-of-neisseria-0JHjq9Anyf
SP - ftx023
VL - 75
IS - 2
DP - DeepDyve
ER -