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Interaction mechanisms of encapsulated meningococci with eucaryotic cells: what does this tell us about the crossing of the blood–brain barrier by Neisseria meningitidis? Xavier Nassif An important feature of Neisseria meningitidis is its ability to invade the meninges. This requires that bacteria cross the blood–brain barrier (BBB), which is one of the tightest barriers of the body. N. meningitidis has, therefore, evolved very sophisticated means by which it circumvents the physical properties of this cellular barrier. Recent advances have allowed the identification of several steps that might occur in the interaction of N. meningitidis with the BBB and the transit of the bacteria to the meninges. Addresses INSERM U411, Laboratoire de Microbiologie, Faculté de Médecine Necker-Enfants Malades, 156 Rue de Vaugirard, 75730 Paris cedex 15, France; e-mail: [email protected] Current Opinion in Microbiology 1999, 2:71–77 http://biomednet.com/elecref/1369527400200071 © Elsevier Science Ltd ISSN 1369-5274 Abbreviations BBB blood–brain barrier CSF cerebrospinal fluid

Introduction Neisseria meningitidis (the meningococcus) is an extracellular pathogen responsible for meningitis and septicaemia. The meningococci colonise the nasopharynx and spread from person to person by droplet infection. In a small percentage of colonised people, the meningococci gain entry into the bloodstream, where it causes meningococcemia and/or progresses to the cerebrospinal fluid (CSF) to cause meningitis after crossing the blood–brain barrier (BBB). In order to reach the meninges from the throat, N. meningitidis has to interact with two cellular barriers: firstly, one in the nasopharynx to invade the bloodstream, and secondly, the BBB in the brain. The BBB is made of two different structures. One structure consists of the endothelium of the brain capillaries, which differ from the endothelial cells present in peripheral capillaries by the presence of tight junctions limiting the paracellular flux [1]. The second structure responsible for the BBB is the choroidal plexus, which is the major site of CSF synthesis and is located in the ventricles [2]. In the choroidal plexus, the endothelial cells are fenestrated and the BBB is formed by tight junctions at the ventricular surface of the epithelial cells. To be able to invade the meninges after colonisation of the throat, meningococci need two kinds of virulence factors: first, attributes responsible for bloodstream survival and dissemination, and second, components mediating the meningococcal interaction with cellular

membranes leading to the bloodstream invasion from the throat, and the crossing of the BBB. Among the former, the importance of the polysaccharide capsule was established by the observation that noncapsulated isolates are usually found in the nasopharynx, whereas bacteria recovered from the blood or the CSF are capsulated. Regarding the latter virulence factors, several bacterial components capable of mediating the interaction of N. meningitidis with cells have been described; however, the sequence of events that take place when meningococci interact with mammalian cells and the events that lead to the crossing of the BBB is far from completely understood. This review discusses the recent developments concerning the bacterial attributes by which virulent N. meningitidis may interact with human cells, and the mechanisms by which the meningococcal cell interactions may lead to the crossing of a cellular barrier such as the BBB.

Meningococcal pili, the indispensable attribute Pili are filamentous structures emanating from the bacterial surface. They are of paramount importance to the pathogenic process, as evidenced by the fact that primary cultures of clinical isolates are always piliated. In vitro, their expression is essential in mediating the interaction between encapsulated meningococci and both epithelial and endothelial cells, because nonpiliated bacteria are unable to adhere and/or to invade these cells [3,4]. In addition to this effect, meningococcal pili have numerous functions. Expression of pili is also associated with a number of other phenotypes, such as a high level competence for transformation by exogenous DNA, bacterial autoagglutination, and twitching motility, which designates a form of bacterial surface translocation. The meningococcal pili belong to the Type IV family of pilins, like the pili of many Gram-negative bacterial pathogens including Neisseria gonorrhoeae, Vibrio cholerae, enterophathogenic Escherichia coli, and Pseudomonas aeruginosa. This family of pilins was originally defined on the basis of related morphologies and polarised localisation on the cells. Studies have shown that this broad classification scheme corresponds well with the presence of a highly conserved amino-terminal domain in the pilin, which is the structural subunit [5]. Evidence for relatedness of these organelles also derives from the conservation of genes and gene products necessary for their expression. This includes prepilins (encoded at the pilE locus in pathogenic Neisseria), prepilin peptidase, soluble proteins with consensus nucleotide-binding motifs and cytoplasmic membrane proteins. In addition,

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homologues corresponding to one or more of these molecules have been implicated in the main terminal branch of the general secretory pathway [6] and expression of competence in Bacillus subtilis [7]. To date, the function of only a few of these molecules, involved in pilus biogenesis, has been studied in depth [8]. Role of pilin and pilin glycosylation

The meningococcal pilin is a well characterised molecule. A single meningococcus cell is genetically capable of producing antigenically different pilin variants. This antigenic variation is due to changes in the primary sequence of the pilE locus. Pilin antigenic variation occurs in vitro [9–11] and in vivo [12]. The genetic basis for pilin antigenic variation has been studied intensively (for reviews see [13,14]). N. meningitidis produces two types of pilins designated class I and class II [15–17]. Both class I and class II pilin-producing strains have been isolated from patients with meningococcal disease. Class I pilins are similar to those of N. gonorrhoeae and the overall organisation of the pilE gene is identical in both species. There is a highly conserved 5′ region encoding amino acids 1–53, which comprises the amino-terminal alpha helix of the protein. This conserved domain is followed by a semivariable region encoding amino acids 54–114. This central region of the molecule contains several highly conserved sequences. The remaining 3′ portion of pilE, encoding the disulphide region and carboxy-terminal tail of pilin, is often referred to as the hypervariable region. Class II pili display a smaller subunit size and the characterisation of the expression locus has only recently been reported [18•]. It shares extensive amino acid identity with the amino-terminal conserved regions of class I meningococcal and gonococcal pilins; however, the sequence displays several unique features compared to the sequences of other neisserial pilin. The crystallographic analysis of one gonococcal pilin variant of strain MS11 reveals a ladle-shaped molecule divided into several distinct structural regions: an amino-terminal hydrophobic alpha helix, a sugar loop carrying an O-linked disaccharide, a four-stranded antiparallel beta-sheet containing a loop connection, and a disulphide loop demarcated by the conserved cystein residues and followed by a carboxyterminal tail [19]. Pathogenic Neisseria pilin has recently been shown to be glycosylated. This modification has been studied using two class I strains. The sugar is O-linked to a Ser that is highly conserved in both class I and class II pilins. Two types of sugar have been found. An O-linked galactose, alpha-1,3 Nacetyl-D-glucosamine (GlcNAc) was first identified in N. gonorrhoeae [19] and then as being the sugar linked to the pilin of one N. meningitidis strain [20•]. In another N. meningitidis strain the sugar identified was a trisaccharide, a digalactosyl 2,4-diacetamido-2,4,6-trideoxyhexose [21]. This suggests that the biological role of glycosylation may not be determined by the precise nature of the

modification, but might be due to a general property conferred by glycosylation independent of the sugar chemistry. Loss of glycosylation does not affect pilus biogenesis. On the contrary, it favours agglutination of pili and the formation of bundles of pili, whereas glycosylation increases the amount of soluble, truncated monomers of pilin [20•]. In addition, the loss of glycosylation is responsible for a slight increase in pilus-mediated adhesion as a consequence of the increase in piliation. The fact that glycosylation increases the amount of soluble pilin monomer suggests that these unassembled pilin molecules could have a function. A cellbinding domain is available in the constant region of pilin monomer [22], which according to the pilin structure would not be available for binding in the pilus fibre. Therefore, pilin as a soluble monomer could signal to cells via this cellbinding domain and have an effect independent of its role as the building block of pili. The pathways that lead to pilin glycosylation remain mostly unknown. Recently a bacterial gene involved in pilin glycosylation has been identified [23••]; this gene probably encodes a galactoside transferase responsible specifically for pilin glycosylation. Other posttranslational modifications have been reported for pilin: a glycerophosphate [24], and a phosphorylcholine epitope are linked to pilin [25•]. The role of these modifications has not been explored yet. Pilin is essential in pilus-mediated adhesion, since pilE– strains are nonpiliated [3,4], however, the data that have been obtained do not support a role for pilin as adhesin. On the other hand, the type of pilin variant expressed by a strain is crucial in determining bacterial adhesiveness. Hence some variants are responsible for a high adhesive phenotype, whereas others result in a low adhesive phenotype [26,27]. Initial studies suggested that this difference might be due to pilin glycosylation [28]. Nonglycosylated derivatives of low and high adhesive variants, however, have been engineered, and still express a similar adhesive phenotype [20•]. On the other hand, it has been clearly shown that the ability to promote high adhesiveness is linked to the formation of bundled pili [22]. Two mechanisms could be responsible for the enhancement of adhesiveness by bundled pili: first, aggregative pili increase bacteria–bacteria interactions, and second, bundled pili could reinforce the interactions of the adhesin with its eucaryotic receptor, because at the extremity of a bundle several molecules of adhesin are present. The mechanism by which some pilins are responsible for pilus aggregation is not known. Comparison of the primary sequence of low and high adhesive derivative suggests that localised charge modifications are responsible for the tendency of certain variants to aggregate [19]. Role of the PilC molecules

The PilC proteins are other important players in both pilus-mediated adhesion, and pilus biogenesis. These proteins, initially described as copurifying with pilin [29], have been found at the tip of the pilus [30] and in the outer membrane where they are believed to be at the

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base of the fibre [31]. Usually meningococcal strains have two pilC loci encoding two proteins designated PilC1 and PilC2, which have 70% identity [3,29,32]. In some strains, the pilC2 locus is missing [33]. These proteins, encoded by two genes pilC1 and pilC2, are subject to phase variation as the result of frameshift mutations within a poly-G tract present in the 5′ end of the genes. These proteins are essential for pilus biogenesis and pilC1–/pilC2– mutants are nonpiliated [3,29]. Furthermore, they function interchangeably in pilus assembly and piliation, because either one of the two alleles is sufficient for pili to be expressed. In addition to this role, PilC1 is required for pilus-mediated adhesion, since PilC1–/PilC2+ isolates are piliated but nonadhesive [3,31], whereas PilC1+ bacteria are adhesive regardless of the PilC2 phenotype. In the light of data obtained with N. gonorrhoeae, the most likely explanation is that PilC1 carries the cell-binding domain [30]. The reason why N. meningitidis strains expressing PilC2 alone are not adhesive is not known, a simple explanation would be that this allele lacks the cell-binding domain.

however, no definite proof for this has been reported. The consequences for the cell of this initial attachment between pili and MCP remain to be assessed. The discovery of a pilus receptor will lead in the near future to a better understanding of the cellular events following pilus-mediated interaction.

Not only one phenotype expressed by these two alleles is different, but their expression is under different regulatory pathways. Hence, the expression of PilC1 is transiently upregulated during the initial contact between the bacteria and the cells, whereas the expression of PilC2 is not affected by the bacteria–cell interaction [34]. This upregulation does not require an initial step of pilus-mediated adhesion, since this regulatory pathway exists in nonpiliated bacteria. This regulatory process is under the control of a pilC1-specific promotor located in a 150 bp DNA region upstream of the gene [35]. The cell contactdependent upregulation of the expression of PilC1 is essential for pilus-mediated adhesion, because its disappearance is responsible for a significant decrease in meningococcal adhesiveness. On the other hand, the cell contact-dependent upregulation of PilC1 does not modify the level of piliation. This suggests that this regulatory pathway is directly linked to the adhesive function of PilC1. A possible explanation for this is that a basal level of PilC1 is sufficient to promote the formation of pili, whereas a higher production may be required to localise PilC1 in the pilus and subsequently at the tip of the fibre. This may explain why, in a recent work, PilC1 of bacteria grown on standard media was found associated only with the outer membrane and not with the pili [31], in contrast to previous reports [30].

Opc and Opa proteins are capable of mediating meningococcal cell interactions only in a noncapsulated background. In capsulated bacteria, Opa and Opc do not seem to affect bacterial interactions with host cells [39–41]. In addition, Opa and Opc mediated cellular interactions are also modulated by lipooigosaccharide (LOS) sialylation. Sialylation of LOS profoundly inhibits bacterial interactions with endothelial and epithelial cells [39,40].

The pilus receptor

The complement regulatory protein (CD46) has recently been recognized as a receptor for piliated meningococci [36••]. Only piliated cells are capable of binding to CHO cells expressing human CD46 and attachment of bacteria could be blocked by monoclonal antibodies against CD46 and by recombinant CD46 protein produced in E. coli. Considering the above model, PilC1 proteins are the best candidates for carrying the binding domain to CD46,

Opa and Opc proteins The Opa (Class 5) proteins are basic outer membrane proteins with a molecular weight of ~28 kDa. Their migration on polyacrylamide gels is heat modifiable [37]. The Opc protein is restricted to a subset of N. meningitidis strains, whereas Opa proteins seem to be widely present in a variety of N. meningitidis strains. A comparison of sequences of Opa variants show that these proteins vary mainly at two hypervariable regions, HV1 and HV2 [13]. Opc also undergoes phase variation, and data suggest that this process may be due to transcriptional regulation [38]. The transcriptional control of Opc may vary from no expression to intermediate expression or to a high level of expression.

Role of Opc

In the absence of sialic acid on the outer membrane, the presence of Opc significantly increased adhesion and invasion of bacteria with both Chang cells and HUVECs when compared to an Opc– variant [41]. Opc influences adherence even in a non-pilated (P–) background and promotes interaction of noncapsulated variants not only with human cells, but also to a lesser degree with cells of animal origin. In addition, Opc mediates interactions only when expressed at high levels [38]. On cultured epithelial cells, evidence has recently been obtained that cell-surface proteoglycans are receptors for Opc [39]. The exact nature of the proteoglycan receptors recognised by these adhesins/invasins, however, is unknown. The interaction between Opc and the endothelial cells is mediated via vitronectin in a trimolecular complex. The Opc-producing meningococci interact with vitronectin and use this molecule to attach to the integrin αvβ3 on the apical surface of the endothelial cells [42]. Thus multiple receptors may exist for Opc, and this raises the question of functional similarities between meningococcal Opc and gonococcal OpaA. Hence, gonococci producing the OpaA (or Opa50) variant use cellular proteoglycans for bacterial adherence to CHO cells [43,44], but require vitronectin to complete the bacterial entry process [45–48]. Thus in certain cell types, a possible involvement of proteoglycan

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receptor in a vitronectin mediated interaction may be necessary for internalisation of Opc producing meningococci. Contrary to pilus-mediated adhesion, the Opc-mediated interaction lead to the internalisation of a high proportion of bacteria inside the cells, at least in some cell types [40]. The signalling pathways that lead to the uptake of the bacterial pathogens have not been identified. In the case of N. gonorrhoeae, however, the Opa-mediated heparan-sulphate peptidoglycan-dependent entry involves the phosphatidylcholine specific phospholipase C and acidic sphingomyelinase [49••]. Role of Opa

Opa proteins allow N. meningitidis interaction with epithelial, endothelial and phagocytic cells. They are apparently more efficient in mediating meningococcal adhesion to and invasion of epithelial cells than with HUVECs [40]. Recent studies indicate that members of the carcinoembryonic antigen (CEA) or CD66 family serve as receptor for meningococcal Opa, promoting adherence and internalisation [50,51]. This interaction between Opa protein and the CD66 family has been more extensively studied with N. gonorrhoeae [52•–55•], where CD66-mediated phagocytosis has been shown to require a Src-like tyrosine kinase and rac1-dependent signalling pathway [56•]. In addition, in N. meningitidis, as in N. gonorrhoeae, a proportion of Opa proteins mediate interaction with epithelial cells that may involve heparan sulphate, this may explain the observed interactions between Opa-producing bacteria and Chang cells, which are believed not to express CD66 [39,40,57]. Recently another possible ligand for Opa proteins has been identified by using the yeast two-hybrid system in N. gonorrhoeae. Williams et al. [58•] have shown that one Opa protein binds pyruvate kinase. Subsequently these authors have demonstrated that intracellular gonococci binds pyruvate kinase and require host pyruvate for growth. A similar function for meningococcal Opa proteins has not yet been reported [58•].

How do these mechanisms relate to the crossing of the BBB? Recent in vivo data have been obtained taking advantage of a case of fulminant meningococcemia where death occurred before antibiotic treatment, at the time when the bacteria were crossing the BBB [33]. At this stage meningococci were found adhering to the endothelial cells of both the choroid plexus and the meninges, thus confirming that N. meningitidis is capable of interacting with the components of the BBB, and that this interaction is likely to be required for CSF invasion. Regarding the route used by meningococci to invade the CSF, the fact that adhesiveness occurred preferentially inside the choroid plexus suggests the crossing of the BBB through this route. The absence of bacteria between the choroidal epithelial cells argues for a crossing directly through the capillaries of the meninges. In addition, expression of the PilC1 adhesin was much higher

in bacteria isolated from cultivation of the CSF in comparison to PilC expression from isolates obtained from the blood. This increased expression of PilC1 correlates in vitro with a higher adhesiveness of CSF-isolated bacteria compared to that of meningococci obtained from the blood. This points to a major role of pilus-mediated adhesion in meningococcal interaction with the BBB. A better understanding of all the steps responsible for the crossing of the BBB by N. meningitidis requires in vitro models. Considering that the main cellular characteristic of the BBB is the existence of tight junctions that limit the paracellular flux either in the meningeal capillaries or in the choroid plexus, the in vitro models used to study this step should be either a monolayer of tight junction forming endothelial cells or a monolayer of choroidal epithelial cells forming tight junctions. Even though considerable progress has been made in our understanding of the BBB, such models using human material are not yet available. This has led to the use of epithelial monolayers of human polarised cells presenting organised tight junctions similar to those responsible for the BBB. Using such a model it is possible to distinguish several steps in the transit of Neisseria [59,60•]. The first step, which corresponds to the first 3–4 hours of infection, is a localised adherence with formation of clumps of bacteria on the apical surface of the monolayer. Microvilli surround these clumps, and have a tendency to form extensions from the epithelial cell surface toward the bacterial cells, thus illustrating local changes in the host cell cytoskeleton. Following this step, bacteria spread onto the surface of the cells as the clumps disappear and are replaced by a monolayer of N. meningitidis covering the cell, thus exhibiting a diffuse adherence. Microvilli disappear and pedestal formations, which correspond to actin polymerisation, are observed with condensation below the bacteria. At this stage, bacteria adhere intimately and firmly to the apical membrane, and in some places are found inside the cells, in a vacuole. It should be pointed out that bacteria are not seen between cells. Crossing of the monolayer starts 8–9 hours after infection, and at 24 hours all infected monolayers have been traversed by the bacteria. Furthermore, this crossing is not responsible for a destruction of the intercellular junctions. The identification of the bacterial attributes involved in all these steps is incomplete. As previously mentioned the role of pilus-mediated adhesion is essential most probably during the first step of localised adherence, and the expression of bundle-forming pili may facilitate the formation of microcolonies by increasing interbacterial interactions. At the stage of diffuse adherence, N. meningitidis have lost their pili. The mechanism involved in this loss of piliation does not correspond to an event of phase variation, since bacteria recovered as cell-associated colony forming units (CFUs) are piliated. There are several possible explanations for this process. Firstly, a crosstalk between bacteria and cells could lead to the down-regulation of genes

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involved in pilus biogenesis. Secondly, pili could be retracted thus bringing into contact the outer membrane of the bacteria and the cellular plasmic membrane.

unknown. The identification of these signalling pathways will be essential in our understanding of the cellular events that lead to meningeal invasion by N. meningitidis.

The loss of piliation following the localised adherence step suggests that pili per se are not involved in the intimate attachment observed during the diffuse adherence. The best candidates for this function would be the Opa/Opc proteins. Several observations, however, argue against the role of these molecules in intimate attachment. Firstly, the Opc protein is not found in all N. meningitidis isolates, and strains that do not have an opc gene can be isolated from the CSF [33]. Furthermore, opc– isolates are capable of intimate attachment [59]. Secondly, using naturally occurring Opa– variants intimate attachment occurs and bacterial cells recovered from these monolayers are still Opa–, suggesting that there is no selection for the Opa+ phenotype during intimate attachment [59]. Thirdly, as mentioned above, these proteins are efficient only in a noncapsulated background, however, an initial interaction mediated by pili could down-regulate expression of capsule and allow these outer membrane proteins to become functional. Other bacterial attributes that have been implicated in mediating a direct interaction with cells could be responsible for this intimate attachment. LOS for instance is able to mediate adherence in vitro [61]. Porins could be involved, by analogy with P.IA porin of N. gonorrhoeae which has been shown to promote gonococcal internalisation [62••]. It should be pointed out that an inoculum of nonpiliated bacteria is incapable of diffuse adherence, suggesting that the initial phase of localised adherence is necessary for the expression of some as yet unidentified attributes that will lead to a diffuse adherence phenotype. Pili could be sensory organs which, once they recognise their receptor, transduce a signal to the bacteria which in turn upregulate the expression of some unidentified component(s) necessary for intimate attachment and expression of the signalling events linked to this step. The loss of piliation could result from the same signalling event.

The mechanisms by which bacteria are internalised and then transcytosed is entirely unknown, as is the vacuolar compartment in which they reside during these events. Recently the neisserial IgA1 protease has been suggested to be involved in the intracellular survival by cleaving the LAMP1 protein, a major integral membrane glycoprotein of late endosomes and lysosomes, and preventing phagolysosomal fusion [63•].

Another open question is which bacterial components are involved in the cytoskeletal modifications and actin polymerisation? From the above model, two distinct signalling events can be distinguished, one responsible for the microvilli extensions and associated with the initial localised adherence, and one responsible for microvilli effacement and formation of actin pedestals, which correlates with the diffuse adherence. In N. gonorrhoeae, the interaction of Opa proteins with either CD66 or heparan-sulphate has been shown to transduce signals that could lead to actin polymerisation. As mentioned above, however, the role of Opa proteins in the crossing of a tight junction forming monolayer by N. meningitidis is not well established. Furthermore, it has recently been shown that attachment of piliated Opa– and Opc– meningococci can elicit actin rearrangement and clustering of tyrosine-phosphorylated proteins [60•]. This suggests that other components beside Class 5 proteins may be able to transduce signalling events to the cells. Whether pilus-mediated adhesion itself is capable of this effect is

Conclusion — why the meningeal tropism? The reasons for the meningeal tropism of N. meningitidis are unclear. None of the bacterial attributes identified as mediating the crossing of a cellular barrier by meningococci are specific to the BBB; they have usually been identified using other cell types other than those belonging to the BBB. Furthermore, meningococcus interacts with most human cell types, and in vivo is seen adhering to most of the endothelial cells of the body. One explanation for the meningeal tropism could be that yet unidentified bacterial attributes may be responsible for the specific interaction with a component of the BBB. These attributes would make the crossing much more efficient in the brain than in the other cell types used in vitro. Alternatively, the interaction between N. meningitidis and the BBB that leads to the CSF invasion may not be specific. The high efficiency with which this bacteria interacts with cells and the high level of bacteremia would result in a generalised adhesion to most of the endothelial cells in the body. In the brain, this nonspecific interaction could be followed by invasion of the CSF, the subsequent meningitis resulting from the lack of cellular defence systems in the CSF.

Acknowledgements Thanks to C Tinsley for a careful reading of this manuscript. The work in the laboratory of X Nassif is supported by INSERM, Université René Descartes Paris 5, the DRET and the Fondation pour la Recherche Médicale.

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18. Aho EL, Botten JW, Hall RJ, Larson MK, Ness JK: Characterization of • a class II expression locus from Neisseria meningitidis: evidence for increased diversity among pilin genes in pathogenic Neisseria species. Infect Immun 1997, 65:2613-2620. This manuscript presents the characterisation of the class II pilE gene of strain FAM18, together with a comparative analysis of gonococcal, meningococcal class I and meningococcal class II pilin genes. 19. Parge HE, Forest KT, Hickey MJ, Christensen DA, Getzoff ED, Tainer JA: Structure of the fibre-forming protein pilin at 2.6 Å resolution. Nature 1995, 378:32-38. 20. Marceau M, Forest K, Béretti J-L, Tainer J, Nassif X: Consequence of • the loss of O-linked glycosylation of meningococcal type IV pilin for piliation and pilus-mediated adhesion. Mol Microbiol 1997, 27:705-715. This is the first report analysing the consequence of the loss of pilin glycosylation on piliation and pilus-mediated adhesion. The data reported eliminate a major role for pilin glycosylation in piliation and subsequent pilus-mediated adhesion, but they demonstrate that glycosylation facilitates solubilisation of pilin monomers and individual pilus fibers. 21. Stimson E, Virji M, Makepeace K, Dell A, Morris HR, Payne G, Saunders JR, Jennings MP, Barker S, Panico M et al.: Meningococcal pilin: a glycoprotein substitued with digalactoside 2,4-diacetamido-2,4,6trideoxyhexose. Mol Microbiol 1995, 17:1201-1214.

31. Rahman M, Kallstrom H, Normark S, Jonsson A: PilC of pathogenic Neisseria is associated with the bacterial cell surface. Mol Microbiol 1997, 25:11-25. 32. Jonsson A-B, Pfeifer J, Normark S: Neisseria gonorrhoeae PilC expression provides a selective mechanism for structural diversity of pili. Proc Natl Acad Sci USA 1992, 89:3204-3208. 33. Pron B, Taha M-K, Rambaud C, Fournet J-C, Pattey N, Monnet J-P, Musilek M, Bérétti J-L, Nassif X: Interaction of Neisseria meningitidis with the components of the blood-brain barrier correlates with an increased expression of PilC. J Infect Dis 1997, 176:1285-1292 34. Taha M-K, Morand P, Pereira Y, Eugène E, Giorgini D, Larribe M, Nassif X: Pilus-mediated adhesion of Neisseria meningitidis — the essential role of cell contact-dependent transcriptional upregulation of the PilC1 protein. Mol Microbiol 1998, 28:1153-1163. 35. Taha M-K, Giorgini D, Nassif X: The pilA regulatory gene modulates the pilus-mediated adhesion of Neisseria meningitidis by controling the transcription of pilC1. Mol Microbiol 1996, 19:1073-1074. 36. Kallstrom H, Liszewski MK, Atkinson JP, Jonsson A-B: Membrane •• cofactor protein (MCP or CD46) is a cellular pilus receptor for pathogenic Neisseria. Mol Microbiol 1997, 25:639-647. This is the first report of the description of a pilus receptor. Anti-CD46 antibodies block the binding of pathogenic Neisseria to epithelial cells. In addition binding to nonhuman cells, such as CHO, can be restored by introducing the human CD46 cDNA. The role of PilC as being the bacterial component binding to CD46 has not been demonstrated. 37.

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22. Marceau M, Beretti J-L, Nassif X: High adhesiveness of encapsulated Neisseria meningitidis to epithelial cells is associated with the formation of bundles of pili. Mol Microbiol 1995, 17:855-863.

38. Sarkari J, Pandt N, Moxon ER, Achtman M: Variable expression of the Opc outer membrane protein in Neisseria meningitidis is caused by size variation of a promoter containing poly-cytidine. Mol Microbiol 1994, 13:207-217.

23. Jennings MP, Virji M, Evans D, Foster V, Srikhanta YN, Steeghs L, •• van der Ley P, Moxon ER: Identification of a novel gene involved in pilin glycosylation in Neisseria meningitidis. Mol Microbiol 1998, 29:975-984. This work reports the identification of a gene designated pglA that encodes a galactoside transferase involved in the addtion of galactose of a trisac-

39. de Vries FP, Cole R, Dankert J, Frosch M, van Putten JPM: Neisseria meningitidis producing the Opc adhesin binds epithelial cell proteoglycan receptors. Mol Microbiol 1998, 27:1203-1212. 40. Virji M, Makepeace K, Ferguson DJP, Achtman M, Moxon ER: Meningococcal Opa and Opc proteins: their role in colonization

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and invasion of human epithelial and endothelial cells. Mol Microbiol 1993, 10:499-510. 41. Virji M, Makepeace K, Ferguson DJP, Achtman M, Sarkari J, Moxon ER: Expression of the Opc protein correlates with invasion of epithelial and endothelial cells by Neisseria meningitidis. Mol Microbiol 1992, 6:2785-2795. 42. Virji M, Makepeace K, Moxon ER: Distinct mechanisms of interactions of Opc-expressing meningococci at apical and basolateral surfaces of human endothelial cells; the role of integrins in apical interactions. Mol Microbiol 1994, 14:173-184. 43. van Putten JP, Paul SM: Binding of syndecan like cell surface proteiglycan receptors is required for Neisseria gonorrhoeae entry into human mucosal cells. EMBO J 1995, 14:2144-2154. 44. Chen T, Belland RJ, Wilson J, Swanson J: Adherence of pilus– Opa+ gonoccoci to epithelial cells in vitro involves heparan sulfate. J Exp Med 1995, 182:511-517. 45. Dehio M, Gomez-Duarte OG, Dehio C, Meyer TF: Vitronectindependent invasion of epithelial cells by Neisseria gonorrhoeae involves ∝v integrin receptors. FEBS Lett 1998, 424:84-88. 46. Duensing TD, van Putten JPM: Vitronectin mediates internalization of Neisseria gonorrhoeae by chinese hamster ovary cells. Infect Immun 1997, 65:964-970. 47.

Gomez-Duarte OG, Dehio M, Guzman CA, Chhatwal GS, Dehio C, Meyer TF: Binding of vitronectin to Opa-expressing Neisseria gonorrhoeae mediates invasion of HeLa cells. Infect Immun 1997, 65:3857-3866.

48. van Putten JPM, Duensing TD, Cole RL: Entry of OpaA+ gonococci into HEp-2 cells requires concerted action of glycosaminoglycans, fibronectin and integrin receptors. Mol Microbiol 1998, 29:369-379. 49. Grassmé H, Gulbins E, Brenner B, Ferlinz K, Sandhoff K, Harzer K, •• Lang F, Meyer TF: Acidic sphingomyelinase mediates entry of N. gonorrhoeae into nonphagocytic cells. Cell 1997, 91:605-615. This manuscript shows that activation of phospholipase C and acidic sphingomyelinase is an essential requirement for the entry of Neisseria gonorrhoeae into nonphagocytic human cell induced by the Opa50 protein. These data provide evidence for a novel function of the acidic sphingomyelinasesignaling pathway. 50. Virji M, Makepeace K, Ferguson DJP, Watt SM: Carconoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors for Opa proteins of pathogenic Neisseria. Mol Microbiol 1996, 22:941-950. 51. Virji M, Watt SM, Barker S, Makepeace K, Doyonnas R: The Ndomain of the human CD66a adhesion molecule is a target for Opa proteins of Neisseria meningitidis and Neisseria gonorrhoeae. Mol Microbiol 1996, 22:929-939. 52. Grayowen SD, Dehio C, Haude A, Grunert F, Meyer TF: CD66 • carcinoembryonic antigens mediate interactions between Opaexpressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes. EMBO J 1997, 16:3435-3445. See annotation for [56•]. 53. Bos M, Grunert F, Belland RJ: Differential recognition of members • of the carcinoembryonic antigen family by Opa variants of Neisseria gonorrhoeae. Infect Immun 1997, 6:2353-2361. See annotation for [56•].

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54. Bos MP, Kuroki M, Krop-Watorek A, Hogan D, Belland RJ: CD66 • receptor specificity exhibited by neisserial Opa variants is controlled by protein determinants in CD66 N-domains. Proc Natl Acad Sci USA 1998, 95:9584-9589. See annotation for [56•]. 55. Chen T, Grunert F, Medina-Marino A, Gotschlich E: Several • carcinoembryonic antigens (CD66) serve as receptors for gonococcal opacity proteins. J Exp Med 1997, 185:1557-1564. See annotation for [56•]. 56. Hauck CR, Meyer TF, Lang F, Gulbins E: CD66-mediated • phagocytosis of Opa52 Neisseria gonorrhoeae requires a Src-like tyrosine kinase- and Rac1-dependent signalling pathway. EMBO J 1998, 17:443-454. This paper, along with [50,51,52•−55•], demonstrates the nice work studying the interaction of Opa proteins with the members of the CD66 family. 57.

de Vries FP, van der Ende A, van Putten JPM, Dankert J: Invasion of primary nasopharyngeal cells by Neisseria meningitidis is controlled by phase variation of multiple surface antigens. Infect Immun 1996, 64:2998-3006.

58. Williams JM, Chen G-C, Zhu L, Rest RF: Using the yeast two-hybrid • system to identify human epithelial cell proteins that bind gonococcal Opa proteins: intracellular gonococci bind pyruvate kinase via their Opa proteins and require host pyruvate for growth. Mol Microbiol 1998, 27:171-186. This work shows that the yeast two-hybrid system is a valuable tool for identifying interactions between bacteria and host proteins. 59. Pujol C, Eugène E, de Saint Martin L, Nassif X: Interaction of Neisseria meningitidis with a polarized monolayer of epithelial cells. Infect Immun 1997, 65:4836-4842. 60. Merz AJ, So M: Attachment of piliated, Opa(-) and Opc(-) • gonococci and meningococci to epithelial cells elicits cortical actin rearrangements and clustering of tyrosine-phosphorylated proteins. Infect Immun 1997, 65:4341-4349. This manuscript demonstrates that piliated Opa –/Opc– pathogenic Neisseria can trigger rearrangements of cortical microfilaments and the accumulation of phosphotyrosine-containing proteins at sites of bacterial contact. Thus demonstrating that neisserial components other than Opa or Opc are involved in transducing signals to the cells. 61. Apicella MA, Zhou D, Lee F, Ketterer M, Porat N, Blake M, Gibson B, Stephens D: The biology of the lipooligosaccharide of the pathogenic Neisseria. In Proceedings of The Ninth International Pathogenic Neisseria Conference. 1994 Sept 36–30: Winchester, England; 1994:10-11. 62. van Putten JPM, Duensing TD, Carlson J: Gonococcal invasion of •• epithelial cells driven by P.IA, a bacterial ion channel with GTP binding properties. J Exp Med 1998, 188:941-952. The neisserial P.I porin is a GTP binding protein that forms a voltage-gated channel that translocates into mammalian cell membranes. In this work, the authors demonstrate that P.I confers invasion of N. gonorrhoeae into Chang epithelial cells and that this event is controlled by GTP. 63. Lin L, Ayala P, Larson J, Mulks M, Fukuda M, Carlsson S, Enns C, • So M: The Neisseria Type 2 IgA1 protease cleaves LAMP1 and promotes survival of bacteria within epithelial cells. Mol Microbiol 1997, 24:1083-1094. This work provides evidences that the IgA1 protease cleave LAMP1, a major integral membrane glycoprotein of late endosomes and lysosomes. This hydrolysis promotes bacterial survival within human epithelial cells.