Project 451966

Whooping cough is a contagious disease of the lungs and airways and continues to be a significant health threat to human, especially infants. The bacterial toxin adenylate cyclase (CyaA) is the key virulence factor produced by the whoop-cough causing agent B. pertussis. To function, CyaA needs to be secreted from bacterial cytosol to the extracellular space and fold into a Ca2+-loaded functional structure. It has been shown that the C-terminal repeats-in-toxin (RTX) domain of CyaA plays critical roles in mediating CyaA toxin activity and virulence of B. pertussis by regulating CyaA's efficient secretion and folding. However, the underlying molecular mechanisms are poorly understood. We propose to use a suite of interdisciplinary approaches, including the state-of-the-art single molecule optical tweezers technique, protein engineering, traditional biophysical techniques, computational modelling and biological assays to understand these key molecular mechanisms. We will elucidate the detailed folding pathways of the RTX domain during secretion, and understand the molecular determinants governing the efficient folding of the RTX domain. We will investigate the molecular mechanism via which the folding signal is transmitted from the C-terminus to the N-terminus of the RTX domain, and develop methods to disrupt the folding signal transmitting pathway to inhibit CyaA toxin activity. We will use computational modeling to screen inhibitors to abolish CyaA toxin activity. These studies will lead to a better understanding of the secretion and folding of CyaA, and help develop new approaches to screen and design virulence inhibitors to combat with B. pertussis and other major pathogens.