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. 2009 May 15;199(10):1479-87.
doi: 10.1086/598217.

The Group B Streptococcal Serine-Rich Repeat 1 Glycoprotein Mediates Penetration of the Blood-Brain Barrier

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Free PMC article

The Group B Streptococcal Serine-Rich Repeat 1 Glycoprotein Mediates Penetration of the Blood-Brain Barrier

Nina M van Sorge et al. J Infect Dis. .
Free PMC article

Abstract

Background: Group B Streptococcus (GBS) is the leading cause of bacterial meningitis in newborn infants. Because GBS is able to invade, survive, and cross the blood-brain barrier, we sought to identify surface-expressed virulence factors that contribute to blood-brain barrier penetration and the pathogenesis of meningitis.

Methods: Targeted deletion and insertional mutants were generated in different GBS clinical isolates. Wild-type and mutant bacteria were analyzed for their capacity to adhere to and invade human brain microvascular endothelial cells (hBMECs) and to penetrate the blood-brain barrier using our model of hematogenous meningitis.

Results: Analysis of a GBS (serotype V) clinical isolate revealed the presence of a surface-anchored serine-rich protein, previously designated serine-rich repeat 1 (Srr-1). GBS Srr-1 is a glycosylated protein with high molecular weight. Deletion of srr1 in NCTC 10/84 resulted in a significant decrease in adherence to and invasion of hBMECs. Additional mutants in other GBS serotypes commonly associated with meningitis showed a similar decrease in hBMEC invasion, compared with parental strains. Finally, in mice, wild-type GBS penetrated the blood-brain barrier and established meningitis more frequently than did the Deltasrr1 mutant strain.

Conclusions: Our data suggest that GBS Srr glycoproteins play an important role in crossing the blood-brain barrier and in the development of streptococcal meningitis.

Figures

Figure 1
Figure 1
(A) Schematic diagram of the GBS srr1 locus. RofA-like is a putative transcriptional regulator, srr1 is a putative cell-surface anchored serine-rich repeat protein, nss is similar to nucleotide sugar synthetases, asp are accessory secretory proteins, Gly and gtf are putative glycosyltransferases and secA2/Y2 is a putative sec translocation system. (B) Protein identity between Srr-1 in serotype V GBS strains NCTC 10/84 (deduced from determined nucleotide sequence) and 2603V/R, and between GBS Srr-1 and GspB homologue in S. gordonii. Alignment was performed using COFFEE-T alignment program. N.d. not determined. SP, signal peptide; SRR1, serine-rich repeat domain 1; NRD, non-repeat domain; SRR2, serine-rich repeat domain 2; CWAD, cell wall anchoring domain.
Figure 2
Figure 2
(A) NCTC 10/84 Srr-1 is cross-reactive with GspB-specific anti-serum. NCTC 10/84 Srr-1 migrates as a > 460 kD protein. Specificity was determined using Δsrr1 mutant and Δsrr1+pSrr1 strains as control for protein band in NCTC 10/84 WT protein lysate, whereas S. gordonii WT and ΔgspB strains were used as positive and negative control for staining with GspB-specific anti-serum, respectively. (B) Detection of carbohydrate modifications on NCTC 10/84 Srr-1 protein using the N-acetylglucosamine-specific lectin WGA. Proteins extracts were separated by gel electrophoresis using 3–8% Tris-acetate gradient gels.
Figure 3
Figure 3
Contribution of Srr-1 to (A) adherence and (B) invasion of hBMEC. (C) Attenuation of invasion is rescued by expressing Srr-1 on expression vector pSrr1 (MOI 1). (D) NCTC 10/84 Srr-1 contributes to invasion in A549 and Hela epithelial cells (MOI 1). Contribution of Srr proteins to (E) adherence and (F) invasion of hBMEC in different GBS serotypes. Serotype III expresses Srr-2, whereas serotypes Ia and Ib express the Srr-1 protein. For adherence, bacteria were enumerated after 30 min incubation, whereas invasion was quantified following 2 h of incubation with hBMEC and 2 h of incubation with penicillin/gentamicin to kill extracellular bacteria. All experiments were repeated at least three times, data from a representative experiment are shown. The error bars indicate 95% confidence intervals of the means of three wells. * p < 0.05, ** p < 0.01, *** p < 0.005.
Figure 4
Figure 4
Deletion of srr1 does not affect survival of GBS in mouse blood at (A) 24 h or (B) 48 h after infection compared to survival of WT bacteria. Line indicates median number of bacteria in the group of ten mice. One square represents one mouse. Filled squares represent mice infected with WT bacteria, open squares represent mice infected with Δsrr1 bacteria. (C) Bacterial counts (CFU) in brains, spleens and blood of mice infected with NCTC 10/84 WT or Δsrr1 mutant bacteria at day 4 after infection. Line indicates median number of bacteria in the group of ten mice. One square represents one mouse. Filled squares represent mice infected with WT bacteria, open squares represent mice infected with Δsrr1 bacteria. * p < 0.05
Figure 5
Figure 5
Histopathology of H&E-stained brain tissues of GBS-infected mice. (A) Represents a sample from a mouse infected with the Δsrr1 mutant showing normal brain histopathology (10x). (B–E) Samples from mice infected with WT GBS show meningeal thickening (B), neutrophil infiltration (C), and tissue destruction (D, E). C and E are inserts at higher magnifications (400x) of B (4x) and D (4x).

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