Autoproteolytic mechanism of CdiA toxin release reconstituted in vitro.

Contact-dependent inhibition (CDI) is a mechanism of interbacterial competition in Gram-negative bacteria. Bacteria that contain CDI systems produce a large, filamentous protein, CdiA, on their cell surfaces. CdiA contains a C-terminal toxin domain that is transported across the outer membranes (OMs) of neighboring bacteria. Once inside a target bacterium, the toxin is released from the CdiA protein via a proteolytic mechanism that has not been well characterized. We have developed an in vitro assay to monitor this toxin release process and have identified several conserved amino acids that play critical roles in the autocatalytic mechanism. Our results indicate that a hydrophobic, membrane-like environment is required for CdiA to fold, and the proteolysis occurs through an asparagine cyclization mechanism. Our in vitro assay thus provides a starting point for analyzing the conformational state of the CdiA protein when it is inserted into a target cell's OM and engaged in transporting the toxin across that membrane. IMPORTANCE: It is challenging to develop new antibiotics capable of killing Gram-negative bacteria because their outer membranes are impermeable to many small molecules. Some Gram-negative bacteria, however, deliver much larger protein toxins through the outer membranes of competing bacteria in their environments using contact-dependent inhibition (CDI) systems. How these toxins traverse the outer membranes of their targets is not well understood. We have therefore developed a method to study the toxin delivery process in a highly simplified system using a fragment of a CDI protein. Our results indicate that the CDI protein assembles into a structure in the target membrane that catalyzes the release of the toxin. This CDI protein fragment enables further studies of the toxin delivery mechanism.