Bsp22 protein polymerization drives dynamic adaptation of the Bordetella type III secretion system injectisome

Many gram-negative bacteria are equipped with a type III secretion system (T3SS) injectisome, enabling the direct translocation of effector proteins from the bacterial cytoplasm into host cells. In the case of Bordetella bronchiseptica, a respiratory pathogen of diverse mammals, the T3SS injectisome exhibits a unique needle tip filament formed by the Bsp22 protein, essential for bacterial persistence in mice. Here, we used B. bronchiseptica and B. pertussis with in-frame insertions of short peptide tags into the Bsp22 to elucidate its formation and characteristics via super-resolution imaging with fluorophore-labeled nanobodies and biarsenic probes. We report that on abiotic surfaces, Bsp22 forms flexible filaments that protrude up to several {micro}m from bacterial cells. In these conditions, Bsp22 filaments grow continuously without any apparent growth control, with Bsp22 subunits being added at the distal end. Remarkably, during host cell infection, the growth of Bsp22 filaments is constrained, accompanied by downregulated Bsp22 protein production. When infecting HeLa cells, some Bsp22 filaments form a short physical bridge between the bacterium and the host cell surface. In nasal epithelium, where B. bronchiseptica colonization is specifically restricted to ciliated cells, Bsp22 filaments align parallel to the cilia, pointing towards the cell apical surface. These results highlight the context-specific and environment-influenced dynamic modulation of the length and orientation of Bordetella needle tip filament, uncovering the adaptability of the Bordetella T3SS injectisome in response to conditions encountered during host cell infection.