The Bordetella Effector BteA Interacts With BopN Through Its C-Terminal Region, Thereby Controlling Its Translocation Into Host Cells.

BteA is a cytotoxic effector translocated into host cells via the type III secretion system (T3SS) of Bordetella. The T3SS gatekeeper protein BopN controls BteA translocation, yet the BteA region mediating this regulation remains unknown. Here, we demonstrate that BteA interacts with BopN through its C-terminal region, and this interaction is required for efficient translocation into host cells. Using a CyaA-based translocation assay, we showed that BteA comprising the N-terminal 448 amino acids (BteA-N448) and BteA with a deletion of amino acids 249-448 (BteA-∆249-448) were translocated in a BopN-dependent manner, whereas BteA comprising the N-terminal 248 amino acids (BteA-N248) was translocated independently of BopN. Pull-down assays confirmed that BteA-N448 and BteA-Δ249-448 physically interact with BopN, whereas BteA-N248 does not. ColabFold-based structure prediction identified candidate interface residues, and subsequent mutagenesis revealed that simultaneous alanine substitution of aspartate 263 (D263) and phenylalanine 340 (F340) abolished BopN-dependent translocation. Our results demonstrated that the amino acid residues D263 and F340 in BteA play important roles in BopN-mediated translocation control. Furthermore, the C-terminal 14-amino-acid region of BteA was strongly suggested to be involved in this regulation. The present study provides new insights into the effector-gatekeeper network that controls T3SS-mediated pathogenicity in Bordetella.