Protein transport across membranes involves complex translocation machineries. Most proteins and toxins exploit the endocytosis machinery and intracellular sorting to gain access to target cell compartments, while few others, such as the adenylate cyclase CyaA toxin, carry their own translocation apparatus. The CyaA toxin is a key virulence factor produced by Bordetella pertussis, the causative agent of whooping cough. Whooping cough is a very contagious vaccine-preventable disease that can have dramatic consequences for persons at risk, like children. Indeed, pertussis is responsible for about 200,000 deaths per year, and most deaths occur in young infants who are either not or incompletely vaccinated. CyaA plays an important role in the early stages of respiratory tract colonization by B. pertussis. The CyaA toxin carries its own translocation machinery, and it is the only known toxin reported to-date that is able to invade cells by a direct translocation of its adenylate cyclase catalytic domain across the plasma membrane, from the extracellular environment to the cytosol of eukaryotic target cells. Once translocated, the catalytic domain is activated by host calmodulin binding and produces supraphysiological levels of cAMP that alter cell physiology, leading ultimately to cell death and subversion of host defense. The CyaA translocation process across the cell plasma membrane remains, however, largely unknown. The objective of this project is to address several key issues regarding the structure and function of CyaA. Currently, the high-resolution three-dimensional structure of the full-length CyaA toxin is unknown, as well as its structural organization and stoichiometry once inserted into membranes, both before and after translocation of the catalytic domain across the plasma membrane. Moreover, the molecular mechanism and the forces involved in the catalytic domain translocation across the plasma membrane have not yet been investigated. Finally, the effect of molecular crowding on the catalytic domain translocation across the plasma membrane and its subsequent folding upon calmodulin-binding, leading to enzymatic activation, are unknown. Taken together, our project aims to solve the structure of CyaA in solution and inserted into membrane, and to characterize its translocation process, using a combination of cutting-edge technologies. From an applied perspective, this knowledge will be instrumental in improving CyaA as antigen delivery vector against cancer cells and bacterial infections, and as well as protective antigen for vaccination against whooping cough.