9 Group B Streptococcus Meningitis

Victor Nizet1 and Kelly S. Doran1,2* 1University of California at San Diego, California, USA; 2San Diego State University, San Diego, USA

9.1 Introduction infections are traditionally divided among two forms: early-onset disease (EoD) and Streptococcus agalactiae (Group B Streptococcus, late-onset disease (LoD). Early-onset in- GBS) is a Gram-positive encapsulated fections are described to occur through the bacterium possessing an array of virulence fi rst 7 days of life, but in fact have a median factors that render it capable of producing onset of only 6–8 h of life, presenting acutely serious disease in susceptible hosts, in with pneumonia and respiratory failure particular the human newborn (Maisey et al., complicated by bloodstream infection and 2008a). Notably, GBS is the leading cause of septicaemia. GBS EoD cases result from meningitis in the neonatal period (Brouwer et ascending infection of the bacterium through al., 2010; Thigpen et al., 2011). Although the placental membranes to initiate infection advances in intensive care management and in utero, or, alternatively, by aspiration of antibiotic therapy have changed GBS infected vaginal fl uids during the birth meningitis from a uniformly fatal disease to a process. Premature, low-birth-weight infants frequently curable one, the overall outcome are at increased risk of developing early- remains unfavourable. Morbidity is high; onset infection, with GBS placental infection 25–50% of surviving infants suff er neuro- itself oft en the critical factor triggering logical sequelae of varying severity, including premature labour. In contrast, GBS LoD cerebral palsy, mental retardation, blindness, occurs in infants up to 7 months of age, with deafness or seizures. more indolent symptom progression related The pathogenesis of neonatal GBS to bacteraemia, absence of lung involvement infection begins with the asymptomatic and a high incidence (~50%) of meningitis colonization of the female genital tract. (Baker and Edwards, 2001). Universal Approximately 20–30% of healthy women screening of pregnant women at 35–37 weeks are colonized with GBS on their vaginal or gestation and intrapartum antibiotic rectal mucosa, and 50–70% of infants born to prophylaxis has resulted in a decline in early- these women will themselves become onset GBS invasive disease in the USA (Phares colonized with the bacterium (Baker and et al., 2008; Van Dyke et al., 2009). However, Edwards, 2001). For the purposes of this treatment has not eliminated the epidemiological classifi cation, neonatal GBS incidence of GBS meningitis, and concern has

*[email protected]

been raised about concurrent increases in fl uid barrier (BCSFB). For the purposes of this non-GBS early-onset bacterial infections, review, the BBB and BCSFB are inter- especially in pre-term infants as a result of changeable concepts with respect to vessel increased antibiotic use (Stoll et al., 2002a,b). endothelial cell penetration by GBS. Furthermore, the occurrence of GBS Disruption of BBB integrity is a hallmark meningitis in older children or adults is more event in the pathophysiology of bacterial commonly appreciated, with an approximate meningitis. This disruption may be due to the 4% increase in total number of cases reported combined eff ect of bacterial entry and between 1997 and 2007 in the USA (Thigpen penetration of brain microvascular endo- et al., 2011). No vaccination strategies are thelial cells (BMECs), direct cellular injury by currently in place to prevent GBS infections, bacterial cytotoxins, and/or activation of host but if ultimately achieved they would be infl ammatory pathways that compromise anticipated to reduce the number of BMEC barrier function. It is apparent that the meningitis cases (Thigpen et al., 2011). Here host immune response is not only incapable we review the current understanding of the of controlling infection within the CNS but pathogenesis of GBS meningitis, highlighting also may be responsible for many adverse important bacterial virulence factors and host events during bacterial meningitis (Tunkel interactions that promote disease progression. and Scheld, 1995). A very complex and integrated series of events involving host cytokines, chemokines, proteolytic enzymes 9.2 Pathophysiology of GBS and oxidants appears to be responsible for Meningitis meningitis-induced brain dysfunction. The development of GBS meningitis progresses The pathophysiology of GBS meningitis through phases including: (i) bloodstream varies according to age of onset. In EoD, survival and the development of bacteraemia; autopsy studies demonstrate litt le or no (ii) direct GBS invasion and disruption of the evidence of leptomeningeal infl ammation, BBB/BCSFB; and (iii) GBS multiplication in despite the presence of abundant bacteria, the CSF-containing subarachnoid and ven- vascular thrombosis and parenchymal tricular spaces, which induces infl ammation haemorrhage (Quirante et al., 1974). By with associated pathophysiological alter- contrast, infants with LoD usually have dif- ations leading to the development of neural fuse purulent arachnoiditis with prominent damage. Brain injury results mainly from involvement of the base of the brain (Berman cerebrovascular involvement leading to and Banker, 1966). Similar age-related cerebral ischaemia, brain oedema, hydro- diff erences in central nervous system (CNS) cephalus and increased ICP. pathology are evident in the infant rat model of invasive disease (Ferrieri et al., 1980). These histopathological diff erences refl ect under- 9.2.1 Bloodstream survival and the development of the host immunological development of bacteraemia response in the immediate neonatal period, with a higher proportion of deaths resulting An association between sustained high-level from overwhelming septicaemia. Clinical and bacteraemia and the development of GBS neuropathological studies have documented meningitis has been suggested in humans and the clear association between bacterial in experimental models of haemato geneous meningitis and brain oedema formation, meningitis (Ferrieri et al., 1980; Doran et al., increased intracranial pressure (ICP), seizure 2002a). This observation implies that GBS activity, arterial and venous cerebral vascular bloodstream survival is an important insults, and other neurological sequelae virulence trait to avoid immune clearance by (Scheld et al., 2002). phagocytic killing by host immune cells, prior To produce meningitis, blood-borne GBS to CNS penetration. Neonates are particularly must typically penetrate the blood–brain prone to invasive disease because of their barrier (BBB) and/or the blood–cerebrospinal quantitative or qualitative defi ciencies in 120 V. Nizet and K.S. Doran

phagocytic cell function, specifi c antibody, or 2007). The β-antigen of C protein binds human the classical and alternative complement IgA antibody (Jerlstrom et al., 1991), and non- pathways. In addition to these newborn host specifi c deposition of IgA on the bacterial susceptibilities, GBS possess a number of surface probably inhibits interactions with virulence determinants that promote blood- complement. Finally, a cell-surface protease, stream survival by thwarting key components CspA, targets host fi brinogen, producing of eff ective opsonophagocytic killing such as adherent fi brin-like cleavage products that complement (Fig. 9.1). For example, the coat the bacterial surface and interfere with surface-anchored GBS β-protein prevents complement-mediated opsonophagocytic opsonophagocytosis by binding short clearance (Harris et al., 2003). consensus repeats found in the middle region The profi le of GBS gene transcription of factor H, a host counter-regulator of changes dramatically during growth in complement (Maruvada et al., 2008), enabling human blood, resulting in an altered cell the unbound active region to block C3b morphology and increased expression of deposition on the bacterial cell surface (Jarva complement regulatory proteins (Santi et al., et al., 2004). In addition, the cell-surface GBS 2007; Mereghett i et al., 2008). The sialylated immunogenic bacterial adhesin (BibA) binds GBS capsular polysaccharide (CPS) represents human C3bp, promoting resistance to one of the most critical factors for limiting the phagocytic killing and contributing to eff ectiveness of host complement and virulence in the mouse model (Santi et al., phagocytic defence. Passage of GBS in animals

Interference with C3 complement function Phagocyte lysis and apoptosis Factor H C3 Fibrinogen C3bp β-protein CspA BibA H2O2 Fibrin-like – O2 fragments Antioxidant GBS defence C3 pigment

SOD Sialic acid in polysaccharide Capsule

β-haemolysin/ cytolysin Bind/sequester Charge repulsion PBP1a (pilus subunit PilB) (Dlt operon) Antimicrobial peptide resistance

Fig. 9.1. Mechanisms of GBS immune evasion. GBS express multiple surface-exposed or secreted factors to evade host immune defences and promote bloodstream survival. The PBP1a and the PilB subunit of GBS pili contribute to antimicrobial peptide resistance. The Dlt operon is responsible for increasing incorporation of D-alanine residues in cell-wall teichoic acids, thereby reducing electronegativity and afﬁ nity for cationic antimicrobial peptides. ScpB, the sialic acid capsule, BibA, -protein and CspA all inhibit host clearance of GBS by interfering with complement components C5a, C3 and C3bp. Superoxide dismutase (SOD) properties of the orange carotenoid pigment shield GBS from killing by phagocyte-generated reactive oxygen species. Alternatively, -haemolysin/cytolysin can boost GBS survival by cytolytic or pro-apoptotic injury to host phagocytes. Group B Streptococcus Meningitis 121

increases capsulation, while serial in vitro carotenoid pigment, a property unique to GBS passage leads to reduced capsule expression among haemolytic streptococci, associated (Hakansson et al., 1988), and strains obtained with the cyl operon encoding the from infants with septicaemia or meningitis β-haemolysin/cytolysin cytotoxin (Spellerberg have increased encapsulation compared with et al., 2000). The free-radical scavenging vaginal colonizing strains (Hakansson et al., properties of this carotenoid neutralize 1987). Thus, it appears that GBS capsule hydrogen peroxide, superoxide, hypochlorite expression is induced during bloodstream and singlet oxygen, and thereby provide a replication and repressed while on mucosal or shield against several elements of phagocyte endothelial cell surfaces, a feature common to ROS killing (Liu et al., 2004). Other GBS factors other meningeal pathogens. Currently, ten that have been linked to survival inside GBS capsular serotypes have been identifi ed phagocytic cells and/or dendritic cells include (Ia, Ib, II–IX) based on the diff erent arrange- CPS (Lemire et al., 2012), a pilin protein ments of four monosaccharides (glucose, (Maisey et al., 2008b) and transcriptional galactose, N-acetylglucosamine and sialic response regulators CovR (Cumley et al., acid) into unique repeating units. Serotype III 2012) and CiaR (Quach et al., 2009), which GBS strains have accounted for a majority of may coordinate expression of acid and stress LoD and meningitis (Baker and Edwards, survival genes. 2001; Tazi et al., 2010), but all serotypes contain Another important host defence a terminal-linked sialic acid bound to mechanism inherent to many immune cells is galactose in an 2  3 linkage (Cieslewicz the production of small cationic antimicrobial et al., 2005). The sialic acid moiety provides peptides (AMPs), such as cathelicidins and antiphagocytic protection by impairing defensins. These peptides are att racted deposition of opsonically active complement electrostatically to negatively charged micro- C3 on the bacterial surface. Isogenic GBS bial cell surfaces, followed by their self- mutants lacking CPS or capsular sialylation assembly to form membrane pores or are more susceptible to neutrophil killing and otherwise disrupt membrane integrity. GBS are less virulent in animal models of infection increase their intrinsic resistance to AMPs by (Campbell et al., 1991; Marques et al., 1992). incorporation of positively charged d-alanine Furthermore, the conserved GBS terminal residues into their cell wall teichoic acids, 2  3 linked sialic acid capsular component thereby reducing surface electronegative is identical to a sugar epitope widely displayed charge and affi nity for the cationic peptides on the surface of all mammalian cells. Thus, (Poyart et al., 2001a). A surface-anchored bacterial surface sialylation may have evolved penicillin-binding protein, PBP1a, enhances to mimic host ‘self’ antigens, allowing GBS to GBS resistance to cathelicidins and defensins, avoid immune detection, manipulate thereby reducing GBS susceptibility to killing phagocyte function and dampen the immune by alveolar macrophages and neutrophils response to GBS infection (Carlin et al., 2007). and promoting bacterial survival in a neonatal When GBS are engulfed and contained rat model of GBS infection (Jones et al., 2007). within the phagosome, a rapid release of toxic Similarly, expression of the pilus backbone reactive oxygen species (ROS) is produced protein PilB and the action of the two- through the phagocyte oxidative burst. component regulator CiaR both render GBS Although GBS do not produce catalase, they more resistant to killing by cathelicidin AMPs are nevertheless able to resist ROS killing and (Maisey et al., 2008b; Quach et al., 2009). survive inside macrophage phagolysosomes (Wilson and Weaver, 1985; Cornacchione et al., 1998; Teixeira et al., 2001). GBS possess an 9.2.2 GBS invasion of the BBB endogenous source of the oxygen-metabolite scavenger glutathione (Wilson and Weaver, Following bloodstream survival, GBS 1985), and the GBS SodA enzyme can interacts directly with BBB endothelium, neutralize superoxide anions (Poyart et al., which can result in bacterial invasion of the 2001b). GBS also produce an orange BBB with subsequent infection of the CNS. 122 V. Nizet and K.S. Doran

This process can result from increased Intracellular invasion (transcytosis) permeability of the BBB and/or the direct invasion of BMECs by the pathogen. GBS enter or ‘invade’ brain endothelium Microbial interaction with the BBB may apically and exit the cell on the basolateral involve crossing the brain endothelium by side, thereby crossing the BBB transcellularly direct intracellular invasion and vacuole (Nizet et al., 1997; Lembo et al., 2010). Electron transit (transcytosis), by passage through the microscopy has demonstrated the presence of intercellular junctional spaces (paracytosis) the meningeal pathogen in membrane-bound or by transport inside another host cell vacuoles within HBMECs (Nizet et al., 1997), (phagocyte-facilitated invasion). With the suggesting the involvement of endocytic availability of in vitro tissue culture models of pathways as well as avoidance of lysosomal human (H)BMECs (Stins et al., 1994; Nizet et fusion for BBB traversal. Further HBMEC al., 1997) and animal models of GBS infection invasion can be blocked by inhibition of actin (Doran et al., 2003; Tazi et al., 2010), signiﬁ cant polymerization, suggesting that GBS trigger progress has been made in identifying and rearrangement of the host cytoskeleton and characterizing the molecular determinants induce their own uptake (Nizet et al., 1997). that promote GBS–BBB interaction (Fig. 9.2). This process may be accomplished, at least in

Neutrophil recruitment

Chemokines GBS factors that promote cellular invasion: (IL-8, CXCL1, CXCL2) ACP, pili, GAPDH, FbsB, β-haemolysin/cytolysin, LTA, Srr proteins FbsA/B PilA Srr1 IagA HvgA Alpha C Fibrinogen, collagen protein ICAM 1 LTA Extracellular Glycosaminoglycan matrix

Cell damage Paracellular Cytoskeletal uncovers novel translocation β-haemolysin/ modulation (mechanism uncertain) cytolysin receptors ZO-1 XX FAK GDP cPLA 2α Rho/Rac

PI3K Paxillin Rho/Rac Pili Lmb GTP PilA Collagen, laminin Basal Cellular adherence Cellular invasion

Fig. 9.2. Mechanisms of GBS penetration of the BBB. Surface-expressed proteins FbsA/B, Srr1, PilA, HvgA, lipoteichoic acid (LTA) and alpha C protein (ACP) mediate GBS binding to host cells and extracellular matrix (ECM) components, such as ﬁ brinogen and collagen. Secreted -haemolysin/ cytolysin promotes GBS invasion, possibly by breaking down host barriers to reveal novel receptors on the basement membrane, such as laminin and collagen, as well as promoting neutrophil inﬂ ux that contributes to barrier disruption. GBS also use glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to activate host plasminogen and degrade the ECM. Intracellular GBS invasion is enhanced by bacterial- dependent cytoskeletal rearrangements triggered by host PI3K/AKT- and FAK-signalling pathways and the Rho family of GTPases. Alternatively, GBS can also disrupt tight junction complexes to cross the barrier by a paracellular route. Several GBS adhesins, including FbsB, pili, LTA and ACP, also contribute to cellular invasion. Group B Streptococcus Meningitis 123

part, by tyrosine phosphorylation of focal (TLR) 2, the data strongly suggest that the adhesion kinase (FAK), which occurs upon att enuated phenotype of the ∆iagA mutant is GBS infection. Phosphorylation of FAK not dependent on TLR2 (Doran et al., 2005). induces its association with PI3K and paxillin, The evidence that the LTA surface polymer an actin fi lament adaptor protein (Shin et al., mediates unique host cell interactions is 2006), and is required for effi cient invasion of consistent with early epidemiological studies. HBMECs by GBS. GBS-infected HBMECs Clinical isolates of GBS from infants with also exhibit increased levels of activated Rho EoD or LoD possess higher quantities of cell- family members RhoA and Rac1. Rho family associated LTA than strains isolated from GTPase inhibitors and dominant-negative mucosal surfaces of asymptomatically expression of RhoA and Rac1 are eff ective in colonized infants (Nealon and Matt ingly, blocking GBS invasion (Shin and Kim, 2006). 1983). Furthermore, longer LTA polymer To elucidate the GBS determinants length is characteristic of isolates from involved in the pathogenesis of meningitis, carriers with invasive GBS disease compared many groups have focused on the with asymptomatic carriers. LTA is common characterization of GBS isolates responsible to all GBS sero- and sequence types, but it for CNS disease. Clinical isolates of serotype remains to be determined if ST-17 clones III GBS, which are over-represented in LoD, contain longer or structurally distinct LTA appear to belong to two distinct evolutionary polymers, which may account for their clusters (Musser et al., 1989), which have now increased virulence. been shown through multilocus sequence More recently, the availability of GBS typing to represent a limited number of clonal genome sequences has enabled the complexes (Jones et al., 2003). Of these clones, identifi cation of genes restricted to the ST-17 sequence type (ST)-17 is strongly associated lineage (Tett elin et al., 2005; Brochet et al., with neonatal meningitis and has been 2006). Mosaic variants were identifi ed at a designated as the hypervirulent clone (Lamy single genomic locus encoding a cell wall- et al., 2006). Screening of a GBS ST-17 mutant anchored protein, with two main variants library revealed a unique requirement for the displaying 38% overall amino acid identity, novel ‘invasion associated gene’, iagA, in BBB namely BibA (Santi et al., 2007), and a second penetration by GBS (Doran et al., 2005). gene to be strictly specifi c to the ST-17 clone Decreased invasion of HBMECs by the GBS (Lamy et al., 2006). This gene, now called ∆iagA mutant in vitro was correlated with a hypervirulent GBS adhesin (HvgA), was reduced risk for development of meningitis shown to be required for GBS hypervirulence and markedly diminished lethality in vivo. (Tazi et al., 2010). GBS strains that express Deletion of iagA did not aff ect other key steps HvgA are more effi cient in gut colonization in the pathogenesis of GBS meningitis, and in crossing the intestinal–blood barrier including bloodstream survival, HBMEC and BBB in neonates, including choroid adherence and intracellular survival. Thus, plexus epithelial cells and brain microvascular the iagA-encoded phenotype of GBS has a endothelium (Tazi et al., 2010). Furthermore, specifi c function in promoting HBMEC heterologous expression of HvgA in non- uptake of the pathogen. The iagA gene adhesive bacteria conferred the ability to encodes an enzyme for the biosynthesis of adhere to intestinal barrier and BBB- diglucosyldiacylglycerol, a membrane glyco- constituting cells. lipid that functions as an anchor for Serotypes Ia, Ib and V are also commonly lipoteichoic acid (LTA), indicating that proper isolated from neonates, children and adult LTA anchoring is important to facilitate patients with meningitis (Phares et al., 2008), penetration of the BBB by GBS (Doran et al., suggesting that other GBS determinants 2005). The host cell receptor for GBS LTA that prevalent among these serotypes are also mediates these interactions has yet to be relevant for the pathogenesis of meningitis. identifi ed. While it is known that LTA is a Proteins targeted for cell surface expression molecule recognized by Toll-like receptor in GBS are predicted to share a C-terminal 124 V. Nizet and K.S. Doran

sequence (L/IPXTG) for sortase recognition and promote the establishment of GBS and anchoring to the Gram-positive cell wall. meningitis (Chang et al., 2011). Impaired host Several cell wall-anchored proteins pro- GAG expression diminished GBS penetration moting GBS BBB penetration have been in the CNS in both murine and Drosophila identifi ed and characterized. In a paradigm- models of GBS infection. GBS interactions shift ing study, it was discovered that GBS with other ECM components also have been express surface-associated pili (Lauer et al., described. GBS mutants lacking the cell wall- 2005). Among the sequenced GBS genomes, anchored fi brinogen-binding protein FbsA two genetic loci encoding pili have been (Schubert et al., 2004), and the laminin- identifi ed, pilus island (PI)-1 and PI-2, the binding protein Lmb (Spellerberg et al., 1999), second existing in one of two variants (PI-2a have reduced ability to adhere to or invade and PI-2b), and not all genomes contain both HBMECs in vitro (Tenenbaum et al., 2005, loci (Rosini et al., 2006). GBS PI-2a includes 2007). Many GBS strains harbour another the genes encoding PilB, an LP(x)TG-motif- fi brinogen-binding protein, FbsB (Gutekunst containing protein that polymerizes to form a et al., 2004), which is secreted and structurally pilus backbone, and accessory pilus proteins unrelated to FbsA. Interestingly, the PilA and PilC that are incorporated in the expression level of FbsA and FbsB in ST-17 pilus (Dramsi et al., 2006). Both PilA and PilB strains correlated to an increased fi brinogen- promote adherence to and invasion of brain binding capacity that may contribute to the endothelium, respectively (Maisey et al., hypervirulence of this lineage (Al Safadi et 2007), and PilA has been implicated in BBB al., 2011). The GBS genome encodes penetration in vivo using a mouse model of homologues to fi bronectin-binding proteins haematogenous GBS meningitis (Banerjee et that contribute to adherence, invasion and al., 2011). Analysis of the PilA protein meningeal infl ammation in other strepto- sequence revealed an integrin I-like domain coccal pathogens (Pracht et al., 2005). Whether resembling the A3 domain of human von or not the proteins function in a similar way Willebrand factor, a molecule known to in GBS remains to be determined. interact with collagens. PilA also binds the Fibrinogen is present in the CNS follow- extracellular matrix (ECM) component ing BBB disruption and vascular damage. collagen, and collagen binding enhanced GBS Furthermore, the interaction of fi brinogen att achment as well as uptake into HBMECs in with integrins and non-integrin receptors a dose-dependent manner (Banerjee et al., expressed on cells of the haematopoietic, 2011). The PilA-collagen complex engages immune and nervous systems can induce 2-β1 integrins on brain endothelium to signalling pathways that regulate infl am- promote bacterial att achment and pro- mation and neurodegenerative functions infl ammatory chemokine release. As a result, involved in CNS disease. Interestingly, recent increased neutrophil infi ltration was studies suggest that adherence to fi brinogen correlated with increased BBB permeability may be a general property of GBS (Dramsi et and higher levels of bacterial CNS penetration al., 2012; Seo et al., 2012) to promote in vivo. This study reveals the deleterious role bloodstream survival and host cell inter- of the neutrophil response to the development actions. An important determinant recently of GBS meningitis, and indicates that the GBS implicated in fi brinogen binding and BBB PilA–BBB interaction is an important interaction are the GBS serine rich repeat molecular event that contributes to disease (Srr) glycoproteins (van Sorge et al., 2009; Seo progression and a detrimental outcome for et al., 2012). Srr proteins have a highly the host. In addition to PilA binding collagen, conserved domain organization, including a other GBS factors interact with various ECM long and specialized signal sequence, two proteins and constituents to promote extensive Srr regions that undergo bacterial–BBB interactions. Recently, the GBS glycosylation, and a typical LP(X)TG cell surface-anchored alpha C protein (APC) was wall anchoring motif. GBS strains carry one shown to interact directly with glucos- of two srr gene alleles, designated srr1 aminoglycans (GAGs) on brain endothelium, (Samen et al., 2007) and srr2 (Seifert et al., Group B Streptococcus Meningitis 125

2006), which are similar in architecture but once present in the CNS to amplify the show only limited homology (< 20% identity). host response and disease progression. Expression of the Srr-2 protein seems to be Alternatively, early molecular interactions of restricted to serotype III and ST-17 strains GBS with the BBB and subsequent barrier (Seifert et al., 2006). Targeted mutagenesis of a disruption may alter cellular polarity. It has GBS Δsrr1 mutant resulted in a marked been demonstrated that GBS is capable of reduction in HMBEC adherence and invasion intercellular transit across an epithelial cell (van Sorge et al., 2009). The srr1 genes in GBS barrier, where the bacterium co-localized serotypes Ia, Ib and V, as well as srr2 in the with junctional protein complexes (Soriani et serotype III ST-17 clone, each contributed to al., 2006). Recent data also indicate that GBS HBMEC invasion in vitro, and Srr-1 promoted infection disrupts tight junctional complexes BBB penetration and the development of GBS in brain endothelium (Kim et al., 2012). An meningitis in a mouse model of haema- overall reduction in the distribution of the togenous meningitis (van Sorge et al., 2009). primary BBB tight junction protein, zona Srr-1 contributes to GBS att achment to occludin (ZO)-1, was observed by immuno- HBMECs via the direct interaction of its fl uorescence during GBS infection. Further binding region (BR) with human fi brinogen evidence demonstrated a decrease in protein (Seo et al., 2012). Studies using recombinant levels of ZO-1 and additional tight junction Srr1-BR established a direct protein inter- protein, occludin, following GBS infection action with the amino acid sequence 283–410 compared with the uninfected control (Kim et of the fi brinogen A chain. Structural al., 2012). Whether these interactions act to predictions indicated that the conformation disrupt tight junctional complexes in brain of Srr1-BR resembles that of other related endothelium and result in a non-polarized bacterial proteins that bind to fi brinogen distribution of proteins on the BBB plasma through a ‘dock, lock and latch’ (DLL) membrane, and/or promote GBS intercellular mechanism (Ponnuraj et al., 2003). The DLL transit across the BBB, remains to be mechanism results when fi brinogen engages investigated. a binding cleft between two domains, N2 and Host factors involved in arachidonic N3. At the ligand ‘dock’, the fl exible acid metabolism also contribute to C-terminal extension of the N3 domain (the penetration of the BBB by GBS (Maruvada et ‘latch’) changes conformation, so that it al., 2011). Pharmacological inhibition and ‘locks’ the ligand in place, and forms a gene deletion demonstrated that host

-strand complex with the N2 domain. cytosolic phospholipase A2 (cPLA2) Deletion of the predicted latch domain of contributes to type III GBS invasion of Srr1-BR abolished the interaction of Srr1-BR HBMEC monolayers and penetration into with fi brinogen. In addition, a mutant GBS the brain in vivo. The mechanism probably strain lacking the Srr-1 latch domain exhibited involves lipoxygenated metabolites of reduced binding to HBMECs, and was arachidonic acid, specifi cally cysteinyl signifi cantly att enuated in an in vivo model of leukotrienes released by cPLA2 as well as meningitis (Seo et al., 2012). Further studies protein kinase C (PKC). GBS penetration −/− are required to determine if similar into the CNS in cPLA2 mice was mechanisms for fi brinogen binding and signifi cantly lower than the penetration of disease progression occur in Srr-2-encoding wild-type mice. However, the magnitudes of −/− strains. bacteraemia were similar between cPLA2 and wild-type mice, suggesting that decreased penetration was not the result of Intercellular invasion (paracytosis) decreased levels of blood-borne bacteria.

The host integrins, ECM components and Interestingly, cPLA2 deletion did not aﬀ ect glycosaminoglycans involved in GBS–BBB GBS penetration into non-brain organs, such interactions all preferentially localize to the as the kidneys and spleen, as similar numbers basolateral surface of polarized endothelium. of bacterial counts were recovered from −/− Thus, GBS may interact with these factors cPLA2 and wild-type mice (Maruvada 126 V. Nizet and K.S. Doran

et al., 2011). The basis for this selective role of It is clear that the GBS β-haemolysin/ host cPLA2 in GBS neurotropism is cytolysin (β-h/c) toxin contributes much to unknown. the observed disease pathology. Haemolysin expression has been shown to directly damage brain endothelial cells (Nizet et al., GBS disruption of the BBB 1997), leptomeninges (meningioma cells) and The host infl ammatory response to GBS astrocytes (Alkuwaity et al., 2012) and contributes signifi cantly to the pathogenesis of primary neurons (Reiss et al., 2011). Infection meningitis and CNS injury. A vascular with wild-type GBS and β-h/c+ cell-free distribution of cortical lesions in neonatal rats extracts induced cell death, whereas chal- with GBS meningitis indicates that lenge with β-h/c-defi cient (β-h/c−) mutant disturbances of cerebral blood fl ow contribute strains and β-h/c− extracts did not. Notably, to neuronal damage (Kim et al., 1995). astrocytes were more sensitive to the Infl ammation of individual brain vessels can cytotoxic eff ects of infection than meningioma lead to focal lesions, whereas diff use cells (Alkuwaity et al., 2012). In neurons, cell- alterations of cerebral blood fl ow cause free extracts of GBS β-h/c toxin induced generalized hypoxic/ischaemic injury and apoptosis in a time- and concentration- cerebral oedema (Kim et al., 1995). GBS induces dependent fashion; electron microscopy of nitric oxide (NO) in brain endothelial cells the neurons showed condensation, shrinkage (Glibetic et al., 2001) and in microglial cells, and partial fragmentation of cells and nuclei resulting in neuronal destruction (Lehnardt et as well as damage to mitochondria (Reiss et al., 2006). Furthermore, arteriolar dysfunction al., 2011). In these studies, GBS β-h/c-induced is associated with the presence of oxygen free cell death could not be prevented by caspase radicals thought to be a by-product of inhibitors, nor was caspase activity detected infi ltrating neutrophils (McKnight et al., 1992). in neurons, consistent with observations in Intraventricular inoculation of newborn other cell types including macrophages. piglets with GBS results in an early sharp rise Haemolysin expression has also been shown in CSF tumour necrosis factor- TNF- to promote the development of meningitis levels, followed shortly by prostaglandin in vivo (Doran et al., 2003; Lembo et al., 2010). release and subarachnoid infl ammation (Ling In a murine model of haematogenous et al., 1995). In the neonatal rat model of meningitis, mice infected with β-h/c− mutants meningitis, TNF- production by astrocytes, exhibited lower mortality and decreased microglia and infi ltrating leucocytes con- brain bacterial counts compared with mice tributes to apoptosis of hippo campal neurons infected with the corresponding wild-type (Bogdan et al., 1997) and further increases in GBS strains (Doran et al., 2003). Similarly, BBB permeability (Kim et al., 1997). Recent mutants that lack the negative repressor of studies have verifi ed the levels of cytokine/ β-h/c, CovR (for control of virulence), chemokine, myelo peroxidase (MPO) activity, exhibited high levels of toxin expression and oxidative stress and disruption of the BBB in an increased ability to penetrate the BBB in the hippocampus and cortex of neonate Wistar vivo (Lembo et al., 2010). Multiple studies rats, following GBS meningitis (Barichello have demonstrated that the lipid dipalmitoyl- et al., 2011). In the neonate brain, the phosphatidylcholine (DPPC) provides pro- hippocampus, mainly, produced higher levels tection against β-h/c-mediated injury in of cytokine/chemokine in the early phase of various host cells (Nizet et al., 1996; Doran et infection, while MPO activity remained al., 2002b; Hensler et al., 2008; Alkuwaity et elevated at 4 days post-infection in both brain al., 2012). DPPC might preserve the host cell structures (Barichello et al., 2011). Interestingly, membrane by providing phospholipid in the neonatal rat, simultaneous intracisternal replacement during pore formation and/or administration of dexa methasone with GBS by direct neutralization by binding to toxin challenge markedly reduced the magnitude of itself. The therapeutic potential of surfactant subarachnoid infl ammation, vasculopathy phospholipids in GBS meningitis requires and neuronal injury (Kim et al., 1995). further study. Group B Streptococcus Meningitis 127

9.3 GBS Activation of a CNS pathogen may result in over-activation of BBB Infl ammatory Response endothelium, leading to increased infl ammation that may com promise BBB integrity or The fi rst comprehensive microarray analysis cause neuronal damage. of the BBB endothelium transcriptional Several GBS factors have been implicated response to a pathogen was examined during in promoting BBB activation. Infection of GBS infection, revealing the induction of a HBMECs with a GBS strain lacking β-h/c specifi c set of 80 genes, which function toxin markedly reduced expression of genes together to orchestrate neutrophil recruit ment, involved in the immune response, while an activation and enhanced survival (Doran et al., unencapsulated strain generally induced 2003). The most highly induced genes, similar or greater expression levels for the interleukin (IL)-8, CXCL1 and CXCL2, all same subset of genes (Doran et al., 2003). belong to the CXC chemokine family, which Neutrophil migration across polar HBMEC acts mainly on cells of neutrophil lineage. IL-8 monolayers was stimulated by GBS and its is the most potent chemotactic factor for β-h/c through a process involving IL-8 and neutrophils because it has a high affi nity for ICAM-1. Furthermore, cell-free bacterial both of the chemokine receptors (CXCR1 and supernatants containing β-h/c activity CXCR2) expressed on neutrophils, and it induced IL-8 release, thus identifying this further stimulates neutrophil respiratory toxin as a principal provocative factor for BBB burst, degranulation and adherence to activation (Doran et al., 2003). In more recent endothelial cells. These chemokines have been studies, additional microarray experiments isolated from the CSF of patients with bacterial have demonstrated that the similar gene meningitis, and IL-8 may be an important profi le in HBMECs is eff ected by CovR biomarker to diff erentiate acute bacterial regulation, which can result in high β-h/c meningitis from aseptic meningitis (Pinto expression (Lembo et al., 2010) and PilA Junior et al., 2011). Other GBS-induced expression (Banerjee et al., 2011). Infection of HBMEC genes related specifi cally to CNS HBMECs in vitro with multiple PilA-defi cient neutrophil recruitment were ICAM-1, which GBS strains resulted in less IL-8 protein when upregulated leads to the enhanced secretion compared with the respective wild- adhesion of neutrophils to the brain type parental strains, and treatment of endothelium, and granulocyte–macrophage HBMECs with recombinant PilA protein colony-stimulating factor (GM–CSF), which induced IL-8 transcription, suggesting that increases neutrophil migration across brain PilA is both necessary and suffi cient to activate endo thelium. Absent during GBS infection of the BBB response (Banerjee et al., 2011). HBMECs was the induction of strong pro- Infection in vivo with the PilA-defi cient strain infl ammatory cytokines, such as TNF- or resulted in delayed mortality, decreased IL-1. These data suggest that the BBB neutrophil infi ltration and bacterial CNS represents much more than a physical barrier dissemination, and less expression of KC, the to GBS, and also performs a sentinel function murine homologue of IL-8 (Banerjee et al., by recognizing the threat of infection and 2011). These results indicate that GBS PilA initiating a CNS-protective innate immune directly promotes IL-8 secretion and response. In the case of blood-borne bacteria, a functional neutrophil signalling pathways in specifi c BMEC gene expression programme vivo, resulting in neutrophil recruitment for neutrophil recruitment and activation is during active GBS infection, which may generated, with the absence of the concurrent function in tandem or concurrently with the production of broader spectrum cytokines β-h/c toxin to promote disease progression. (e.g. TNF-, IL-1) that could provoke a wider These fi ndings also demonstrate an or unchecked patt ern of infl ammatory association between leucocyte traffi cking and activation potentially harmful to critical CNS BBB permeability and increased GBS structures. However, the timing and penetration of the CNS, suggesting that magnitude of the neutrophil recruitment polymorphonuclear leucocyte (PMN)- response is critical for the outcome of infection. mediated damage of the BBB has a signifi cant Continued exposure and invasion of the role in the pathogenesis of GBS meningitis. 128 V. Nizet and K.S. Doran

9.4 Conclusions RgfA/C activates the fbsB gene encoding major fi brinogen-binding protein in highly virulent Advances in microbial genetics, tissue culture CC17 clone Group B Streptococcus. PLoS One 6, e14658. systems and small-animal challenge models Alkuwaity, K., Taylor, A., Heckels, J.E., Doran, K.S. have enhanced our understanding of the and Christodoulides, M. (2012) Group B molecular pathogenesis of GBS meningitis Strepto coccus interactions with human and the host response to this potentially life- meningeal cells and astrocytes in vitro. PLoS threatening infection. New model systems One 7, e42660. using zebrafi sh (Patt erson et al., 2012) and Baker, C.J. and Edwards, M.S. (2001) Infectious Drosophila (Baron et al., 2009; Chang et al., Diseases of the Fetus and Newborn Infant. W.B. 2011) promise the contribution of host Saunders, Philadelphia, PA. genetics to enrich our understanding of host– Banerjee, A., Kim, B.J., Carmona, E.M., Cutting, GBS interactions. Comparative genomic and A.S., Gurney, M.A., Carlos, C., Feuer, R., Prasadarao, N.V. and Doran, K.S. (2011) systems-level bioinformatics studies have Bacterial pili exploit integrin machinery to revealed strain evolution associated with promote immune activation and effi cient blood– hypervirulence and CNS disease potential, brain barrier penetration. Nature Com- including specifi c candidate gene and munications 2, 462. regulatory systems that promote bloodstream Barichello, T., Lemos, J.C., Generoso, J.S., survival, HBMEC interactions and activation Cipriano, A.L., Milioli, G.L., Marcelino, D.M., of host infl ammatory responses. Genomics Vuolo, F., Petronilho, F., Dal-Pizzol, F., Vilela, has led to the development of reverse and M.C. and Teixeira, A.L. (2011) Oxidative stress, structural vaccinology technologies for cytokine/chemokine and disruption of blood– vaccine discovery, including a type 2a pilus brain barrier in neonate rats after meningitis by Streptococcus agalactiae. Neurochemical (BP-2a)-based GBS experimental vaccine Research 36, 1922–1930. (Nuccitelli et al., 2011). In addition, genomics Baron, M.J., Wong, S.L., Nybakken, K., Carey, V.J. has led to the discovery of component and Madoff, L.C. (2009) Host glycosaminoglycan proteins and virulence factors as potential confers susceptibility to bacterial infection in vaccine targets. Enhanced understanding of Drosophila melanogaster. Infection and the molecular basis of GBS meningitis may Immunity 77, 860–866. highlight novel bacterial and host molecules Berman, P.H. and Banker, B.Q. (1966) Neonatal as therapeutic or immuno-prophylactic meningitis. A clinical and pathological study of targets against this dangerous infectious 29 cases. Pediatrics 38, 6–24. disease condition of the neonate. Bogdan, I., Leib, S.L., Bergeron, M., Chow, L. and Tauber, M.G. (1997) Tumor necrosis factor- contributes to apoptosis in hippocampal neurons during experimental group B Acknowledgements streptococcal meningitis. Journal of Infectious Diseases 176, 693–697. The authors thank their respective laboratory Brochet, M., Couve, E., Zouine, M., Vallaeys, T., members and researchers whose work has Rusniok, C., Lamy, M.C., Buchrieser, C., Trieu- not been discussed in detail or reviewed Cuot, P., Kunst, F., Poyart, C. and Glaser, P. elsewhere. Work on the BBB and GBS (2006) Genomic diversity and evolution within meningitis in K.S. Doran’s laboratory is the species Streptococcus agalactiae. Microbes supported by funding from the NIH/NINDS and Infection 8, 1227–1243. Brouwer, M.C., Tunkel, A.R. and van de Beek, D. (grant no RO1NS051247). (2010) Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clinical Microbiology Reviews 23, 467–492. Campbell, J.R., Baker, C.J. and Edwards, M.S. References (1991) Deposition and degradation of C3 on type III group B streptococci. Infection and Al Safadi, R., Mereghetti, L., Salloum, M., Lartigue, Immunity 59, 1978–1983. M.F., Virlogeux-Payant, I., Quentin, R. and Carlin, A.F., Lewis, A.L., Varki, A. and Nizet, V. Rosenau, A. (2011) Two-component system (2007) Group B streptococcal capsular sialic Group B Streptococcus Meningitis 129

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