Discovery of pertussis toxin receptors

Bordetella pertussis is the causative agent of pertussis, also known as whooping cough. Among the virulence factors produced by B. pertussis, the ABs toxin called pertussis toxin (PT) is strongly linked to disease symptoms and severity. Like other AB5 toxins, the PT holotoxin has five B subunits that recognize cell surface molecules and one A subunit that harbors an ADP-ribosylation activity. The nonulosonic acid N-acetylneuraminic acid (NeusAc) is commonly described to be the receptor for PT but it is unknown how other glycan features affect PT binding. Additionally, PT has been reported to bind to non-sialylated glycoconjugates. This proposal aims to define the glycoconjugate features that mediate PT recognition and intoxication of lymphocyte and respiratory epithelial cell surfaces, two cell types that are proposed to be targets of PT action in vivo. In Aim 1, CRISPR/Casg genome-wide knockout (KO) screening will be used to identify genes that impact PT binding to cell surfaces. Based on the results of these screens, individual KO cell lines will be constructed and assayed for PT binding, PT internalization, and PT intoxication. KO cell lines will also be evaluated for changes in glycosylation, to reveal which glycan structures are associated with susceptibility to PT. In Aim 2, photocrosslinking sugar technology will be used to identify protein component of glycoproteins that interact directly with PT. Based on the results of this analysis, KO cell lines will be constructed and assayed for PT binding, PT internalization, and PT intoxication. Together, the two aims will reveal the glycan and protein components of PT receptors. This information will explain how PT targets specific cell types and may suggest strategies to interfere with PT action therapeutically. Project Summary/Abstract