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bioRxiv · 10.1101/2023.09.06.556475

Bordetella Filamentous hemagglutinin (FhaB) and Adenylyl cyclase toxin (ACT) interactions on the bacterial surface are consistent with FhaB-mediated delivery of ACT to phagocytic cells

Nash, Z. M.·Inatsuka, C. S.·Cotter, P. A.·Johnson, R. M.
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Abstract

Bordetella species that cause respiratory infections in mammals include B. pertussis, which causes human whooping cough, and B. bronchiseptica, which infects nearly all mammals. Both bacterial species produce filamentous hemagglutinin (FhaB) and adenylyl cyclase toxin (ACT), prominent surface-associated and secreted virulence factors that contribute to persistence in the lower respiratory tract by inhibiting clearance by phagocytic cells. FhaB and ACT proteins interact with themselves, each other, and host cells. Using immunoblot analyses, we showed that ACT binds to FhaB preferentially on the bacterial surface before being released into the extracellular environment. We showed that SphB1, a surface protease identified based on its ability to cleave FhaB, also cleaves ACT, and we showed that the presence of ACT blocks SphB1-dependent and independent cleavage of FhaB, but the presence of FhaB does not block SphB1-dependent cleavage of ACT. SphB1-dependent cleavage of ACT occurs proximally to ACTs active site, and hence is predicted to inactivate ACT. We also showed that FhaB-bound ACT can intoxicate host cells producing CR3, the receptor for ACT. In addition to increasing our understanding of FhaB, ACT, and ACT interactions on the Bordetella surface, our data support a model in which FhaB functions as a novel toxin delivery system by binding to ACT and allowing its release upon binding of ACT to its receptor, CR3, on phagocytic cells. AUTHOR SUMMARYBacteria need to control the variety, abundance, and conformation of proteins on the cellular surface to survive. Members of the Gram-negative bacterial genus Bordetella include B. pertussis, which causes whooping cough in humans, and B. bronchiseptica, which causes respiratory infections in a broad range of mammals. These extremely closely related species produce two prominent virulence factors, the two-partner secretion (TPS) effector FhaB and Adenylyl cyclase toxin (ACT), that interact with themselves, each other, and host cells. In this work, we showed that ACT binds preferentially to FhaB on the bacterial surface before being released into the extracellular environment. We showed that the exoprotease SphB1, which cleaves FhaB, also cleaves ACT. We showed that ACT that is bound to FhaB can be delivered to CR3+ host cells. Our data support a model in which FhaB functions to deliver ACT specifically to phagocytic cells, and not epithelial cells. This is the first report of a TPS system facilitating delivery of a separate polypeptide toxin to target cells and expands our understanding of how these systems contribute to bacterial pathogenesis.

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Nash, Z. M., Inatsuka, C. S., Cotter, P. A., Johnson, R. M.. 2023-09-06. Bordetella Filamentous hemagglutinin (FhaB) and Adenylyl cyclase toxin (ACT) interactions on the bacterial surface are consistent with FhaB-mediated delivery of ACT to phagocytic cells. https://doi.org/10.1101/2023.09.06.556475

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