IDENTIFICATION AND CHARACTERIZATION OF SULFATED GLYCOCONJUGATE RECEPTORS FOR THE BORDETELLA PERTUSSIS ADHESIN FILAMENTOUS HEMAGGLUTININ

Abstract

Filamentous hemagglutinin (FHA) is a major antigen present on the outer membrane surface of Bordetella pertussis, the Gram-negative bacterium that causes the disease pertussis. Active immunization with this protein provides protection against pertussis in animal models of disease. As a component of acellular vaccines under study in humans, FHA has also been shown to play a role in protection against human infection and disease. It has been well documented that FHA functions as a multi-faceted adhesin: this molecule contains an arginine-glycine-aspartic acid (RGD) sequence which binds to the CR3 integrin on the macrophage surface; FHA also interacts with the ciliated epithelium in a manner that does not involve the RGD sequence but which appears to be galactose inhibitable. The interaction of FHA with nonciliated epithelial cells defines a third mechanism of binding with host cells, and it is this mechanism that is explored in this thesis. FHA is the principle adhesin involved in attachment to nonciliated epithelial cells such as WiDr adenocarcinoma cells. In this thesis, it is shown that the Chinese hamster ovary (CHO) epithelial cell line interacts with FHA in a manner that is inhibitable by sulfated polysaccharides, and that FHA binds directly with the sulfated sugar heparin. In addition, it is determined that virulent B. pertussis can bind to galactosylceramide-sulfate (sulfatide) by thin layer chromatography overlay assays, and that this binding is completely inhibitable by dextran sulfate. FHA appears to be the adhesin responsible for binding to sulfatide since it is shown to directly interact with this glycosphingolipid, which is present in large quantities in the upper respiratory epithelium. In order to identify additional potential receptors for FHA on the epithelial cell surface, a CHO cell line deficient in the expression of glycosaminoglycans is shown to exhibit greatly diminished attachment to FHA. Detergent extracts of wild type CHO and HeLa cells contain a sulfated and glycosylated high molecular weight material that binds with high affinity to an FHA-Affigel column, and this material can be efficiently removed from the FHA column with sulfated polysaccharides. Digestion with glycosidases reveals that this material is heparan sulfate glycosaminoglycan. This polysaccharide is covalently linked to core proteins to form proteoglycan macromolecules which are present on the surface of virtually all eukaryotic cells. These studies extend the observation that FHA contains a sulfated polysaccharide binding domain by defining two potential receptors or FHA on the epithelial cell surface, a sulfated glycosphingolipid and heparan sulfate glycosaminoglycan. These eukaryotic molecules are accessible to this bacterium as it first enters the upper respiratory tract, and understanding initial colonization events that may involve binding to these receptors can help to further our understanding of B. pertussis pathogenesis and disease prevention.