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Bordetela peinussis is bound to glycocoijugates on human cilia and macrophages by multiple adhesins, including pertussis toxin. The cellular recognition properties of the B oligomer of pertussis toxin were characterized and the location and structural requirements of the recognition domains were identified by site-directed mutagenesis of recombinant pertussis toxin subunits. Differential recognition of cilia and macrophages, respectively, was localized to subunits S2 and S3 of the B oligomer. Despite >80% sequence homology between these subunits, ciliary lactosylceramide exclusively recognized S2 and leukocytic gangliosides bound only S3. Substitution at residue 44, 45, 50, or 51 in S2 resulted in a shift of carbohydrate recognition from lactosylceramide to gangliosides. Mutational exchange of amino acid residues 37-52 between S2 and S3 interchanged their carbohydrate and target cell specificity. Comparison of these carbohydrate recognition sequences to those of plant and animal lectins revealed that regions essential for function of the prokaryotic lectins were strongly related to a subset of eukaryotic carbohydrate recognition domains of the C type.During the clinical course of whooping cough, Bordetella pertussis specifically attaches to the cilia of the respiratory epithelium (1), establishes an intracellular state within alveolar macrophages (2), and produces systemic disease by elaborating several toxins (3). At least two adhesins, filamentous hemagglutinin and pertussis toxin (PT), are known to mediate bacterial attachment to human cells (4, 5). Both adhesins are required for attachment to cilia, but each can act independently in the adherence of the organism to macrophages. These adhesins have several unusual features of interest. They are large, nonfimbrial molecules that contain multiple binding domains (1, 5) and, when shed from the cell surface, retain the ability to mediate adherence even for other bacterial species (6).PT is a major virulence determinant of B. pertussis, which induces metabolic changes in the host (7), alters immune responses (8), and is the only toxin known to bind whole bacteria to eukaryotic cells (4). The toxin is a hexameric protein with an A-B architecture (9). The A protomer is composed of a single S1 subunit (Mr, 26,026) containing the catalytic site for toxic ADP-ribosylation of cellular signaltransducing guanine nucleotide regulatory proteins. The B oligomer possesses the cellular recognition domains; it is a complex pentamer containing subunits S2 (Me,21,925), S3 (Mr,21,873), S4 (Mr,12,059), and S5 (Mr, 11,013) in a respective molar ratio of 1:1:2:1 (9). By interacting with glycoproteins and glycolipids on many types of eukaryotic cells (4,(10)(11)(12)(13)(14)(15)(16), the B oligomer serves several distinct and independent functions (4, 15): mitogenicity for T lymphocytes, adherence of the bacteria to eukaryotic cells, and delivery of the toxic S1 subunit to its target. Several findings suggest that the B oligomer contains at least two distinct binding specificit...

Pertussis toxin (PTX) is a major virulence factor in whooping cough and can elicit protective antibodies. Amino acid residues 8 to 15 of PTX subunit S1 are important for the adenosine diphosphate-ribosyltransferase activity associated with the pathobiological effects of PTX. Furthermore, this region contains at least a portion of an epitope that elicits both toxin-neutralizing and protective antibody responses in mice. The gene encoding the S1 subunit was subjected to site-specific mutagenesis in this critical region. A mutant containing a single amino acid substitution (Arg9----Lys) had reduced enzymatic activity (approximately 0.02% of control) while retaining the protective epitope. This analog S1 molecule may provide the basis for a genetically detoxified PTX with potential for use as a component of an acellular vaccine against whooping cough.

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