IDENTIFICATION OF A CARBOHYDRATE-RECOGNITION DOMAIN IN FILAMENTOUS HEMAGGLUTININ FROM BORDETELLA-PERTUSSIS

The adherence of Bordetella pertussis to ciliated cells and macrophage s is critical to colonization and infection of the respiratory tract. Adherence to both types of cells involves the recognition of eukaryoti c carbohydrates by the bacterial adhesin filamentous hemagglutinin (Fh a). The carbohydrate recognition domain (CRD) of Fha is considered an important antigen for subcomponent vaccines to maximize the generation of antiadherence antibodies capable of protecting against colonizatio n. For identification of the CRD of Fha, a bank of eight monoclonal an tibodies (MAbs) that mapped to four contiguous regions were tested for their ability to block Fha binding to lactosylceramide or to block ba cterial binding to ciliated cells. Only MAb 12.5A9, which maps to amin o acid residues 1141 to 1279, blocked both Fha binding to lactosylcera mide and bacterial binding to ciliated cells. An 18-kDa polypeptide co rresponding to this region was expressed in Escherichia coli. Cell lys ates containing this protein bound to lactosylceramide in a manner ide ntical to that of native Fha. Mutant strains of B. pertussis that cont ained an in-frame deletion of the coding sequence for this region prod uced a truncated Fha that showed negligible cross-reactivity with MAb 12.5A9. In an adherence assay, these mutant strains failed to bind eff iciently to either ciliated cells or macrophages. The numbers of adher ent bacteria for these strains were reduced to the number obtained wit h a nonadherent strain. We conclude that the region defined by residue s 1141 to 1279 of Fha constitutes a CRD critical for bacterial adheren ce and represents a potential candidate for a subcomponent vaccine.