Group B Streptococcal Pilus Proteins Contribute to Adherence to and Invasion of Brain Microvascular Endothelial Cells

Abstract

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...

Rga is a regulator of adherence and pilus formation in Streptococcus agalactiae

“…Although important data have been published on their biogenesis and their function (Abbot et al, 2007;Dramsi et al, 2006;Gianfaldoni et al, 2007;Lauer et al, 2005;Maisey et al, 2007;Manetti et al, 2007;Mora et al, 2005;Rosini et al, 2006;Scott & Zähner, 2006;Telford et al, 2006), little is known about the regulation of their synthesis. Besides RogB (Dramsi et al, 2006;Gutekunst et al, 2003) SrtA has been described as a regulator of pilus biosynthesis (Dramsi et al, 2006).…”

“…The pilA gene is the first gene of the pil operon encoding a pilus apparatus, which is a multisubunit hairlike structure that extends from the bacterial cell surface (Telford et al, 2006). In S. agalactiae, these structures are involved in adherence to host tissues (Dramsi et al, 2006;Maisey et al, 2007). As Rga activates the transcription of both srr-1 and pilA, and thereby increases the amount of Srr-1 and pilus proteins on the surface of the bacteria, it is consistent that Rga significantly increases bacterial adherence to some host cells.…”

“…Srr-1, PilA and FbsA were previously reported to be involved in the bacterial adherence to host cells (Maisey et al, 2007;Samen et al, 2007;Schubert et al, 2004). In particular, it was previously demonstrated that Srr-1 is involved in the adherence of S. agalactiae to the human larynx cell line HEp-2, and that FbsA mediates the binding to human pulmonary cell line A549 (Samen et al, 2007;Schubert et al, 2004).…”

“…PilA of S. agalactiae is a component of pili structures on the surface of the bacteria (Dramsi et al, 2006;Maisey et al, 2007;Rosini et al, 2006). Following SDS-PAGE with gradient polyacrylamide gels, pilus structures form a ladder of bands corresponding to high-molecular-mass proteins (Dramsi et al, 2006;Gianfaldoni et al, 2007;Lauer et al, 2005;Mora et al, 2005;Rosini et al, 2006).…”

Sortase D Forms the Covalent Bond That Links BcpB to the Tip of Bacillus cereus Pili

“…Minor pilin subunits are critically important for bacterial adherence to host cell surfaces, which is also a prerequisite for invasion or biofilm formation (24 -27). For example, the minor pilin subunit of Streptococcus agalactiae provides for bacterial adherence to the brain endothelium as well as pulmonary epithelial cells (28,29). Corynebacterium diphtheriae assemble pili from three subunits, the major pilin SpaA, and two minor pilins, SpaC and SpaB (14).…”