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Penetration of the Blood Brain Barrier in GBS Meningitis

Investigators
Nizet, Victor
Institutions
University of California - San Diego
Start date
1999
End date
2001
Objective
In this application for the NICHD Small Grants Program, we propose to utilize molecular genetic techniques and our tissue culture model to identify and characterize GBS genes involved in BMEC invasion. This will be accomplished by (a) screening a transposon Tn917 mutant library of wild-type III GBS strain COH1 to identify mutants deficient in BMEC invasion and (b) screening a library of chromosomal DNA cloned into the noninvasive S. gordonii for recombinants plasmids which confer BMEC-invasive ability.
More information
Group B streptococci (GBS) are the leading cause of meningitis in newborn infants. Although meningitis develops following bacteremia, the precise mechanism(s) whereby GBS leave the bloodstream and gain access to the central nervous system (CNS) are not known. We hypothesized that GBS produce meningitis because of a unique capacity to invade human brain microvascular endothelial cells (BMEC), the single cell layer which constitutes the blood-brain barrier. We developed an in vitro tissue culture model to test this hypothesis and have published our initial findings. GBS are able to efficiently invade and survive within BMEC, apparently by eliciting their own endocytotic uptake. GBS invasion of BMEC requires active bacterial DNA, RNA, and protein synthesis. Serotype III strains, which account for the large majority of CNS isolates, invade BMEC more efficiently than strains from other common GBS serotypes, and are capable of transcytosis across polar monolayers of BMEC. In pilot studies, we have confirmed that GBS localize to BMEC as they penetrate the blood brain barrier in vivo. We now feel prepared to test the hypothesis that specific GBS genes and gene products are responsible for the ability of GBS to invade BMEC, cross the blood-brain barrier and produce neonatal meningitis. In this application for the NICHD Small Grants Program, we propose to utilize molecular genetic techniques and our tissue culture model to identify and characterize GBS genes involved in BMEC invasion. This will be accomplished by (a) screening a transposon Tn917 mutant library of wild-type III GBS strain COH1 to identify mutants deficient in BMEC invasion and (b) screening a library of chromosomal DNA cloned into the noninvasive S. gordonii for recombinants plasmids which confer BMEC-invasive ability. Identification of putative invasion genes will be accomplished by sequence analysis of Tn917 insertion sites associated with a noninvasive phenotype or cloned DNA fragments which confer invasive ability to S. gordonii. This sequence information will be used to construct targeted BMEC invasion gene knockouts by homologous recombination-based plasmid integrational mutagenesis, an independent confirmation of the importance of the gene(s) to the BMEC-invasive phenotype. Finally, using the specific BMEC invasion gene knockouts, we propose to verify the requirement of BMEC invasion in the pathogenesis of blood-brain barrier penetration and GBS meningitis using an infant rat model.
Funding Source
Nat'l. Inst. of Child Health and Human Devel.
Project number
5R03HD037224-02
Categories
Sanitation and Quality Standards