Binding of pertussis toxin (PTx) was examined by glycan microarray; 53 positive hits fell into four general groups. One group represents sialylated bi-antennary compounds with an N-glycan core terminating in α2-6 linked sialic acid. The second group consists of multi-antennary compounds with an N-glycan core, but lacking terminal sialic acids, which represents a departure from previous understanding of PTx binding. The third group consists of Neu5Acα2-3Lactose that lack the branched-mannose core found in N-glycans, thus their presentation is more similar to O-glycans and glycolipids. The fourth group of compounds consists of Neu5Acα2-8Neu5Acα2-8Neu5Ac, which is seen in the c-series gangliosides and some N-glycans. Quantitative analysis by SPR of the relative affinities of PTx for terminal Neu5Acα2-3 versus Neu5Acα2-6, as well as the affinities for the trisaccharide Neu5Acα2-8Neu5Acα2-8Neu5Ac versus disaccharide, revealed identical global affinities, even though the amount of bound glycan varied by 4- to 5-fold. These studies suggest that the conformational space occupied by a glycan can play an important role in binding, independent of affinity. Characterization of N-terminal and C-terminal binding sites on the S2 and S3 subunits by mutational analysis revealed that binding to all sialylated compounds is mediated by the C-terminal binding sites, and binding to non-sialylated N-linked glycans is mediated by the N-terminal sites present on both the S2 and S3 subunits. An understanding of the glycans recognized by PTx is essential to understand which cells are targeted in clinical disease.

In addition to delivering S1 into target cells, the B-pentamer has toxic effects independent of S1, including antigen-independent T-cell activation and mitogenicity. Flow cytometry and microscopic analysis also revealed PTx-B promotes membrane exchange. Jurkat cells stained with the lipophylic dye DiI transferred DiI to DiO stained cells, and vice versa. In addition, the intracellular amine-coupled fluorophor was also transferred, suggesting that protein transfer also occurred. T-cells take up plasma membrane and integral membrane proteins from antigen presenting cells during immunological synapse contact, a process called trogocytosis. Unlike trogocytosis, PTx-B mediated transfer does not require direct cell-to-cell contact. While T-cell activation by PTx-B requires the TCR complex, PTx-B mediated membrane transfer occurred in TCR-deficient mutants. Mitogenic lectins, ConA, PHA-L, and WGA mediated aggregation, but only PTx-B and WGA mediated membrane transfer. Actin inhibitors, Cytochalasin-D and Latrunculin-B, inhibited both the transfer of membrane material and aggregation, suggesting a role for cytoskeletal remodeling. The activity of S2S4 and S3S4 dimers was also tested. Both dimers activated TCR signaling and induced aggregation, but only the S2S4 dimer mediated membrane exchange. Mutant analysis revealed that the sialic acid binding site was required for TCR signaling for both dimers, as well as membrane exchange by the S2S4 dimer. The membrane transfer process may contribute to disregulation of immune responses by PTx since proteins transferred in an immunological synapse have been shown to retain signaling activity, and loss of adhesion molecules may contribute to the alterations of immune cell trafficking. Widespread and improper immune activation or immune cell trafficking by PTx-B may aid the ability of B. pertussis to reinfect immune hosts.