Group B streptococci (GBSs) are the leading cause of neonatal meningitis. GBSs enter the CNS by penetrating the blood-brain barrier (BBB), which consists of specialized human brain microvascular endothelial cells (hBMECs). To identify GBS factors required for BBB penetration, we generated random mutant libraries of a virulent strain and screened for loss of hBMEC invasion in vitro. Two independent hypo-invasive mutants possessed disruptions in the same gene, invasion associated gene (iagA), which encodes a glycosyltransferase homolog. Allelic replacement of iagA in the GBS chromosome produced a 4-fold decrease in hBMEC invasiveness. Mice challenged with the GBS ∆iagA mutant developed bacteremia comparably to WT mice, yet mortality was significantly lower (20% vs. 90%), as was the incidence of meningitis. The glycolipid diglucosyldiacylglycerol, a cell membrane anchor for lipoteichoic acid (LTA) and predicted product of the IagA glycosyltransferase, was absent in the ∆iagA mutant, which consequently shed LTA into the media. Attenuation of virulence of the ∆iagA mutant was found to be independent of TLR2-mediated signaling, but bacterial supernatants from the ∆iagA mutant containing released LTA inhibited hBMEC invasion by WT GBS. Our data suggest that LTA expression on the GBS surface plays a role in bacterial interaction with BBB endothelium and the pathogenesis of neonatal meningitis.

Surface filamentous structures known as pili have been discovered recently in the gram-positive streptococcal pathogens that cause invasive disease in humans, including group B Streptococcus (GBS). We show that two GBS proteins involved in pilus formation, encoded by pilA and pilB, also facilitate the interaction of this important agent of central nervous system infection with endothelial cells of the human blood-brain barrier.Group B Streptococcus (GBS), a gram-positive bacterial pathogen, is a major cause of meningitis in newborns. In order to cause central nervous system infection, blood-borne GBS must interact with and breech the blood-brain barrier (BBB), comprised primarily of a single layer of brain microvascular endothelial cells. While GBS adheres to and invades human brain microvascular endothelial cells (hBMEC) (9), the specific GBS factors that contribute to this process are only beginning to be elucidated. Proper anchoring of lipoteichoic acid on the GBS surface facilitates hBMEC invasion, while the pore-forming ␤-hemolysin/cytolysin is cytolytic for hBMEC; each factor promotes BBB penetration and lethality in a mouse model of hematogenous meningitis (1, 2). A recent study has also demonstrated that GBS fibrinogen-binding protein FbsA contributes to hBMEC adherence and invasion in vitro (14).Proteins targeted for cell surface expression in GBS are predicted to share a C-terminal sequence (L/IPXTG) for sortase recognition and anchoring to the gram-positive cell wall, and we have examined candidate genes encoding this motif for roles in the cellular pathogenesis of GBS meningitis. Serotype V GBS clinical isolate NCTC10/84 is highly virulent in the mouse model of hematogenous meningitis (2), and our PCR and sequence analysis of this strain revealed a locus encoding the transcriptional regulator RogB (5), three genes encoding proteins with C-terminal L/IPXTG anchor motifs, and two candidate sortase enzyme genes (Fig. 1A). Compared to loci present in the sequenced GBS strains NEM316 (4) and 2603V/R (15), in which the corresponding L/IPXTG-anchored proteins have been demonstrated to be components of pilus appendages (3, 10), this locus exhibited a high level of sequence identity and had a similar position in the chromosome. Comparison of our locus with the previous reports suggested that the second L/IPXTG-anchored protein (encoded by a gene designated pilB) could represent the major pilus subunit. Homologues of other pilus genes at a second pilus locus found in several other GBS strains (10) were not detected in the NCTC10/84 strain.GBS pilus components, including the PilB homologue GBS59, were shown to induce protective immunity in mouse models of GBS disease and thus show promise for use in the development of novel vaccines (8, 10). Given the exposure of these components on the bacterial surface, we hypothesized that pilus expression by GBS could also play a role in the bacterium's initial interactions with hBMEC. Utilizing a molecular genetic approach that combined targeted allelic replacement and het...

Group B Streptococcus commonly colonises healthy adults without symptoms, yet under certain circumstances displays the ability to invade host tissues, evade immune detection and cause serious invasive disease. Consequently, Group B Streptococcus remains a leading cause of neonatal pneumonia, sepsis and meningitis. Here we review recent information on the bacterial factors and mechanisms that direct host-pathogen interactions involved in the pathogenesis of Group B Streptococcus infection. New research on host signalling and inflammatory responses to Group B Streptococcus infection is summarised. An understanding of the complex interplay between Group B Streptococcus and host provides valuable insight into pathogen evolution and highlights molecular targets for therapeutic intervention.GBS (Group B Streptococcus/-cocci) is a leading agent of severe, invasive bacterial infection in human newborns. Neonatal infection with this opportunistic pathogen can present as earlyonset or late-onset disease. In early-onset cases, bacteria are transferred from the mother to the infant in utero, following ascending infection of the placental membranes, or during passage through the birth canal, by aspiration of infected vaginal fluids. Early-onset neonatal infection manifests within the first few hours or days of life, often presenting as pneumonia and respiratory failure, which can quickly progress to bacteraemia and septic shock. By contrast, late-onset GBS disease can occur in infants up to several months old, and is distinguished by bloodstream infection with a high rate (40-60%) of progression to meningitis (Ref. 1). Infants that survive GBS meningitis can suffer serious long-term neurological consequences, such as seizures, hearing loss and cognitive impairment. Serious GBS infections are increasingly recognised in adult populations, particularly in the elderly and individuals compromised by underlying medical conditions. More than 40% of all invasive GBS cases in the USA occur past infancy (Ref. 2).The development of GBS disease reflects successful bacterial colonisation of the vaginal epithelium, penetration of placental or epithelial barriers, resistance to immune clearance allowing bloodstream survival and, in cases of meningitis, the ability to breach the endothelial blood-brain barrier (BBB). In overcoming these obstacles, GBS expresses a diverse array of surface-associated and secreted virulence factors that mediate specific host-cell interactions and interfere with innate immune clearance mechanisms. The present review explores

Group B Streptococcus (GBS) is a major cause of invasive bacterial infections in newborns and certain adult populations. Surface filamentous appendages known as pili have been recently identified in GBS. However, little is known about the role of these structures in disease pathogenesis. In this study we sought to probe potential functional role(s) of PilB, the major GBS pilus protein subunit, by coupling analysis of an isogenic GBS pilB knockout strain with heterologous expression of the pilB gene in the nonpathogenic bacterium Lactococcus lactis. We found the knockout GBS strain that lacked PilB was more susceptible than wild-type (WT) GBS to killing by isolated macrophages and neutrophils. Survival was linked to the ability of PilB to mediate GBS resistance to cathelicidin antimi-crobial peptides. Furthermore, the PilB-deficient GBS mutant was more readily cleared from the mouse bloodstream and less-virulent in vivo compared to the WT parent strain. Strikingly, overexpression of the pilB gene alone in L. lactis enhanced resistance to phagocyte killing, increased bloodstream survival, and conferred virulence in a mouse challenge model. Together these data demonstrate that the pilus backbone subunit, PilB, plays an integral role in GBS virulence and suggests a novel role for gram-positive pili in thwarting the innate defenses of phagocyte killing. Keywords Streptococcus agalactiae; pili; GBS; macrophage; neutrophil; antimicrobial peptidesThe gram-positive bacterium Group B Streptococcus (GBS) is a major cause of pneumonia, sepsis, and meningitis in newborns and is increasingly associated with disease in adult populations including the elderly, pregnant women, and diabetics (1). Cell surface-expressed NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript filamentous appendages known as pili have recently been identified in streptococcal pathogens that cause invasive infections in humans, including GBS (2), Group A Streptococcus (GAS) (3), and Streptococcus pneumoniae (4). Pili, also known as fimbriae, are non-flagellar polymeric organelles often involved in bacterial adherence to host cells and tissues during colonization. The analysis of multiple GBS genomes has revealed the presence of specific genetic islands that contain the necessary components for pilus formation (5). In all cases that have been described so far, the genes that encode the pilus proteins are clustered at the same genetic locus, transcribed in the same direction, and are likely part of an operon (6). The pilus operon codes for three proteins with the conserved C-terminal amino acid motif LP(X)TG for subsequent cell wall anchoring, and two genes encoding sortases required for complete pilus assembly. Recent studies have demonstrated that a single GBS pilin protein constitutes the major pilus subunit or bona fide pilus, while ancillary proteins are incorporated at the tip or base of the pilus structure (5,7,8). In addition, an immediate upstream transcriptional regulator has been shown to activate the expression of...

Group A Streptococcus is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Cell wall anchored pili were recently described in several species of pathogenic streptococci, and in the case of GAS, these surface appendages were demonstrated to facilitate epithelial cell adherence. Here we use targeted mutagenesis to evaluate the contribution of pilus expression to virulence of the globally disseminated M1T1 GAS clone, the leading agent of both GAS pharyngitis and severe invasive infections. We confirm that pilus expression promotes GAS adherence to pharyngeal cells, keratinocytes, and skin. However, in contrast to findings reported for group B streptococcal and pneumococcal pili, we observe that pilus expression reduces GAS virulence in murine models of necrotizing fasciitis, pneumonia and sepsis, while decreasing GAS survival in human blood. Further analysis indicated the systemic virulence attenuation associated with pilus expression was not related to differences in phagocytic uptake, complement deposition or cathelicidin antimicrobial peptide sensitivity. Rather, GAS pili were found to induce neutrophil IL-8 production, promote neutrophil transcytosis of endothelial cells, and increase neutrophil release of DNA-based extracellular traps, ultimately promoting GAS entrapment and killing within these structures.