GBS are the leading cause of meningitis in human newborns.  While all serotypes may produce meningitis, capsule serotype III strains account for ~80% of cases.  Although neonatal meningitis develops as a consequence of hematogenous spread of the organism, the factors responsible for GBS entry into the central nervous system (CNS) have not been determined.  The blood-brain barrier (BBB) consists of a single layer of specialized brain microvascular endothelial cells (BMEC) which exhibit continuous tight junctions and are responsible for maintaining biochemical homeostasis within the CNS.  We had observed GBS organisms inside the BMEC of neonatal rats infected intraperitoneally and sacrificed histopathologic examination of brain tissue.  We hypothesized that GBS produce meningitis because of a unique capacity to invade human brain microvascular endothelial cells.  To test this hypothesis, we developed together with Prof. Kwang Sik Kim (now at Johns Hopkins) an in vitro model of GBS blood-brain barrier interactions using BMEC isolated from a human, immortalized by SV40 transformation, and propagated in tissue culture monolayers.  GBS invasion of BMEC was demonstrated by electron microscopy, the first ever photographic evidence that a human meningitis pathogen could penetrate the cells which constitute the blood-brain barrier.  Intracellular GBS were found within membrane-bound vacuoles, suggest the organism induced its own endocytotic uptake. 

We adapted a gentamicin protection assay to quantify intracellular GBS.  Serotype III strains, which account for the majority of CNS isolates, invaded BMEC more efficiently that strains from other serotypes.  GBS invasion required active  bacterial DNA, RNA and protein synthesis, as well as microfilament and microtubule elements of the eukaryotic cytoskeleton.  At high bacterial densities, GBS invasion of BMEC was accompanied by evidence of cellular injury, which in turn was correlated to hemolysin/cytolysin production by the organism.  Finally, we demonstrated GBS transcytosis across intact, polar BMEC monolayers grown on Transwell membranes.  We concluded that GBS invasion of BMEC is a primary step in the pathogenesis of neonatal meningitis, allowing bacteria access to the CNS by transcytosis or by injury and disruption of the endothelial BBB.  Recently we used transposon mutagenesis to discover a gene, iagA, required for GBS BBB invasion in vitro and in vivo.  The iagA gene was found to encode for the glycolipid anchor for lipoteichoic acid to the GBS cell membrane, identifying an novel function for lipoteichoic acid in GBS cellular invasion and disease pathogenesis.

Ongoing studies led by our colleague Dr. Kelly Doran employ genetic techniques including transposon mutagenesis and heterologous expression to identify specific GBS genes and gene products responsible for GBS invasion of BMEC.  We have also utilized high density oligonucleotide microarrays to identify host genes important in the initial inflammatory response of the BBB to a bacterial pathogen.  We found that GBS infection induced a highly specific and coordinate set of genes known to orchestrate neutrophil recruitment, activation and enhanced survival.  Prominent induced genes included the C-X-C family chemokines interleukin (IL)-8, Gro-alpha and Gro-beta, along with IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), myeloid cell leukemia sequence 1 (Mcl-1) and intercellular adhesion molecule 1 (ICAM-1).  Key findings were confirmed by Real Time RT-PCR and immunoassays.  Specific bacterial triggers for BBB gene activation were sought by parallel studies using isogenic mutants lacking important GBS virulence factors. Aß-h/c deficient strain exhibited a significantly reduced ability to induce the same subset of genes, identifying it as the principal provocative factor for BBB activation.  In contrast, an unencapsulated strain induced greater expression of several genes, suggesting the capsule may act as a “cloak” to diminish host recognition of the pathogen.  It appears the innate immune response of BBB endothelium to GBS is to summon circulating neutrophils, a response that is modulated by specific bacterial virulence determinants.  

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