Genome-wide interaction screen for Mycobacterium tuberculosis ClpCP protease reveals toxin-antitoxin systems as a major substrate class.

In Mycobacterium tuberculosis (Mtb), the Clp protease degradation pathway, mediated by the modular ClpCP and ClpXP protease complexes, is essential for growth and presents an attractive drug target. Employing a bacterial adenylate cyclase two-hybrid (BACTH) screening approach that we adapted to screen the proteome of an Mtb ORF library, we identify protein interaction partners of the ClpC1 chaperone on a genome-wide level. Our results demonstrate that bipartite type II toxin-antitoxin (TA) systems represent a major substrate class. Out of the 67 type II TA systems known in Mtb, 25 appear as ClpC1 interaction partners in the BACTH screen, including members of the VapBC, MazEF, and ParDE families, as well as a RelBE member that was identified biochemically. We show that antitoxins of the Vap and Rel families are degraded by ClpCP in vitro. We also demonstrate that ClpCP is responsible for mediating the N-end rule pathway, since the adaptor protein ClpS supports ClpC-dependent degradation of an N-end rule model substrate in vitro.