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Group B Streptococcal entry into brain endothelial cells
Givens, Brandon A.
Group B Streptococcus (GBS) is a Gram-positive bacterial pathogen that has been associated with the development of neonatal disease, namely meningitis. GBS has the ability to persist in the bloodstream and ultimately cross the blood-brain barrier (BBB). Previous research has proven that GBS can directly enter human brain microvascular endothelial cells (hBMEC), but the exact mechanism of cellular entry is not known. It has been reported that various microbes utilize clathrin- or caveolin-mediated endocytosis or lipid rafts for entry into host cells. Therefore we assessed GBS invasion in hBMEC in the presence of specific inhibitors of endocytosis. Our results demonstrate that inhibition of clathrin-mediated endocytosis, through the disruption of the essential protein crosslinking reactions, resulted in decreased bacterial invasion. However bacterial uptake was not completely abated, suggesting that additional entry mechanisms may be involved. This prompted us to examine other endocytic mechanisms. We assessed the level of GBS uptake into hBMEC in the presence of inhibitors of caveolin-mediated endocytosis and lipid raft formation. Our results demonstrated that inhibition of the caveolae pathway through the use of the inhibitor genistein, resulted in a significant decrease in bacterial invasion. Additionally disruption of lipid raft formation through the removal of cholesterol by Methyl-β-cyclodextrin also produced similar results. The clathrin binding protein ATG16L1 also may play a role in GBS uptake as GBS uptake into an ATG16L1 KO cell line was greatly reduced. In order to look at possible GBS factors that may interact with these pathways we analyzed the ability of various GBS mutant strains, deficient in cell surface/adhesion proteins, to invade hBMEC in the presence of pathway inhibitors. We found that capsulation is important for GBS recognition via lipid rafts capitulating results in other cell lines. Invasion mutants with knock outs in srr2 or iagA observed no differences when compared to wildtype. As a whole our data suggests that caveolin/lipid raft mediated endocytosis along with clathrin-mediated entry is important for GBS invasion into the brain endothelium. We hope that these findings will help further the understanding of GBS disruption of the BBB and the progression of meningeal disease.
Master of Science (M.S.) San Diego State University, 2017
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