Balamuthia amoebic encephalitis (BAE) is a serious human disease almost always leading to death. An important step in BAE is amoebae invasion of the bloodstream, followed by their haematogenous spread. Balamuthia mandrillaris entry into the central nervous system most likely occurs at the blood–brain barrier sites. Using human brain microvascular endothelial cells (HBMECs), which constitute the blood–brain barrier, this study determined (i) the ability of B. mandrillaris to bind to HBMECs and (ii) the associated molecular mechanisms. Adhesion assays revealed that B. mandrillaris exhibited greater than 90 % binding to HBMECs in vitro. To determine whether recognition of carbohydrate moieties on the surface of the HBMECs plays a role in B. mandrillaris adherence to the target cells, adhesion assays were performed in the presence of the saccharides mannose, galactose, xylose, glucose and fucose. It was observed that adherence of B. mandrillaris was significantly reduced by galactose, whilst the other saccharides had no effect. Acetone fixation of amoebae, but not of HBMECs, abolished adhesion, suggesting that B. mandrillaris adhesin(s) bind to galactose-containing glycoproteins of HBMECs. B. mandrillaris also bound to microtitre wells coated with galactose–BSA. By affinity chromatography using a galactose–Sepharose column, a galactose-binding protein (GBP) was isolated from detergent extracts of unlabelled amoebae. The isolation of a GBP from cell-surface-biotin-labelled amoebae suggested its membrane association. One-dimensional SDS-PAGE confirmed the proteinaceous nature of the GBP and determined its molecular mass as approximately 100 kDa. This is the first report suggesting the role of a GBP in B. mandrillaris interactions with HBMECs.
Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier
