Abstract 1128 Catalytic Roles of Key Amino Acids in the Toxin Cleavage Mechanism of a Bacterial Inhibition Protein

Some Gram-negative bacteria produce contact-dependent inhibition (CDI) systems that deliver protein toxins into neighboring competing cells. A critical component of CDI systems is CdiA, a cell surface protein that contains a toxin domain at its C-terminal end. Upon binding to a receptor protein on a target cell, CdiA transfers the toxin domain across the target's outer membrane. The toxin is subsequently cleaved from CdiA by a process that is believed to be autoproteolytic. We have developed an in vitro assay to monitor the cleavage of a purified CdiA fragment in detergent solution. Our assay establishes that the toxin release is catalyzed by the folding of the CdiA protein into a hydrophobic environment and that several conserved amino acids are involved in the proteolytic mechanism. Through mutational analysis, we have identified the catalytic roles of several of these amino acids. Our results suggest that the cleavage proceeds through an intein-like asparagine cyclization mechanism. We would like to thank the College of the Holy Cross and the Cottrell Scholar Award #28179 sponsored by Research Corporation for Science Advancement for their valuable support in making our project a reality.