Deciphering the role of BscX and BscY subunits in the Bordetella Type 3 Secretion System

Monika Cizkova1,2, Vaclav Veverka3, Peter Sebo1, Jana Kamanova1, Ladislav Bumba1

1Institute of Microbiology AS CR v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic

2Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, Prague 6, Czech Republic

3Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic

The type 3 secretion system (T3SS), also known as an injectosome, is a multisubunit protein-export apparatus that enables the delivery of bacterial effector proteins directly from bacterial cytosol into the cytosol of the host cells. Structural organization of the injectosomes is highly homologous among the Gram-negative bacteria, but some additional regulatory and structural components are species specific. In Bordetella pertussis, the causative agent of whooping cough, as well as in Bordetella bronchiseptica, which primarily causes respiratory infections of rodents, these include small protein subunits, BscX and BscY, of which the structure and function remain unknown. Here, we present the solution structure of the BscX-BscY heterodimer determined by nuclear magnetic resonance spectroscopy and demonstrate that both BscX and BscY subunits are critical for the proper function of the T3SS apparatus. The structure shows that BscY adopts a six helical fold, which is wrapped by BscX consisting of the long N-terminal unstructured region with three helices at the C‑terminus of the protein. In vitro pull-down assays revealed that the BscX-BscY heterodimer directly interacts with the cytosolic domain of the BcrD protein, suggesting that the heterodimer might regulate the interaction of the inner membrane export apparatus with the cytoplasmic sorting platform. Even though both subunits appear to be tightly connected within the complex, the BscX subunit was found to be secreted out of the bacterial cells, demonstrating certain dynamics of the T3SS apparatus during the secretion of the effectors. Moreover, deletion of the individual bscX and bscY genes, or the removal of the N-terminal twelve residues of BscX rendered the bacterial mutant non-cytotoxic against the HeLa cells, indicating that the structural integrity of the BscX and BscY subunits is essential for the proper function of the Bordetella T3SS apparatus.